Microfluidic devices

ABSTRACT

A microfluidic device configured to sample, meter and collect a metered volume of body fluid for analysis by means of capillary transport, comprises: an inlet section, for receiving a sample of body fluid, the inlet section comprising an inlet port; a metering section configured to receive body fluid from the inlet section and comprising a metering channel, wherein the metering section is arranged to separate a metered volume of body fluid filled in the metering channel; and an outlet section configured to receive and transport the separated metered volume of body fluid for collection in a capillary means having a predetermined surface geometry. The metering channel has an outlet part with a dimensional change configured to cause a fluid front meniscus of the separated metered volume of body fluid, when transported to the outlet section, to assume a shape which substantially conforms to the surface geometry of the capillary means.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationSerial No. PCT/SE2022/050645, filed on Jun. 28, 2022, which claimspriority to Swedish Patent Application No. 2150835-3, filed Jun. 29,2021, and Swedish Patent Application No. 2150836-1, filed Jun. 29, 2021,which are hereby incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates generally to microfluidic plasmaextraction and metering thereof from whole blood, specifically to amicrofluidic device configured to sample and collect a metered volume ofbody fluid for analysis by means of capillary transport, comprising afiltration membrane configured to separate selected cells from the bodyfluid and extract the body fluid.

BACKGROUND ART

Separation of plasma from whole blood is a key step within whole-bloodtesting for clinical diagnostics and biomedical research purposes. Bloodsampling is conventionally done through venipuncture and collection of5-10 ml of whole blood in a tube. For analysis, plasma is usually thepreferred substance; it is obtained through centrifugation in acentralized laboratory prior to analysis. An alternative collectionmethod to handling liquid samples in tubes, is to apply the blood on apaper material and allow the sample to dry in on the paper. In thelaboratory, the dried blood can be re-dissolved and prepared foranalysis through wet chemistry. This method is called Dried Blood Spotanalysis (DBS) and when combined with a separation technology forretaining blood cells, one can also obtain Dried Plasma Spots (DPS).This methodology has gained popularity as it brings the advantage of norequirement for maintaining a cold chain during transportation to thelab. The simplicity of the storage format also opens up for capillaryhome sampling by finger prick.

Microfluidic systems and Lab-on-Chips are solutions for reducing timeand cost of biochemical assays. Through miniaturization, the volumes tobe analyzed are reduced which shortens reaction times and reduces theconsumption of expensive reagents amongst others. Microfluidictechnology has been applied for plasma extraction purposes. Separationof blood cells from plasma on the microscale can be achieved eitheractively (externally applied force such as electrical or magnetic field)or passively (sedimentation, filtration or hydrodynamic effects inducedby microfeatures). Further paper-based, and centrifugal microfluidicsalso can be applied.

For example, US 2014/0332098 A1 discloses circuit elements forself-powered, self-regulating microfluidic circuits includingprogrammable retention valves, programmable trigger valves, enhancedcapillary pumps, and flow resonators. Some embodiments allow for theflow direction within a microfluidic circuit to be reversed as well asfor retention of reagents prior to sale or deployment of themicrofluidic circuit for eased user use.

Many biochemical analyses require quantitation of analytes. To determinethe precise concentration of an analyte in a sample, knowledge of theprecise sample volume is required. On a microfluidic level, metering ofliquids can again be achieved actively or passively. Examples of activemeans of dividing a volume of fluid into two or more volumes are byintroducing components such as active valves that mechanically interferewith the liquid volume to split it up in units or passive valves incombination with pressurized air that can tear off parts of a liquid. Indroplet microfluidics, shear forces that appears between two immiscibleliquid phases (oil and water) in certain microfluidic geometries(T-junctions) are exploited for liquid compartmentalization. Passivemetering has been reported less frequently in the literature. WO2016/209147 A1 demonstrates passive metering using two dissolvablemembranes integrated in a microchannel. Further, US 2015/0147777 A1 usesintersecting overspill channel structures containing absorbing materialsfor metering. WO 2015/044454 A2 discloses a microfluidic device forcollecting and transporting biofluids, preferably whole blood, andincludes a slope and a metering channel for collecting a metered sample.This device has a first region with a low flow resistance, comprisinginlet features, and a second region comprising the metering channel witha high flow resistance, which is an arrangement that may cause problemsrelated to obtaining a stable performance adapted to different flowsresulting from variations in blood characteristics.

It is desirable to enable completely autonomous systems for plasmasampling. Such an autonomous system for plasma sampling has theadvantage of requiring minimal interaction from the user running theprocess, thereby allowing a reduced training level of the user and areduced risk of errors during sampling. An autonomous system by passivemeans on a microfluidic level would further reduce the complexity andcost of the system, as no external driving forces requiring powersources etc. would be required to run the microfluidic functions.However, developing such a system would involve substantial designchallenges, such as making the system tolerate a wide range of wholeblood characteristics in terms of varying hematocrit, lipid content andcoagulation factors which vary largely between individuals, becausethese variances generate differences in flow characteristics in thesystem which would be easier to manipulate by active flow manipulation.The present disclosure is directed to improvements that solves thementioned problems, while resulting in a volume defined plasma sample.

One aspect of the problems to be addressed in the microfluidic deviceinvolves microfluidics, specifically, how to generate a height gradientin a microfluidic substrate. The fabrication of microfluidic channelswith a gradient in channel height seldomly occurs in research or inindustrial microfluidic applications due to the difficulty infabricating slants or slopes on microfluidic substrates. Slants may beproduced through CNC micro milling, electroplating or 3D printing. Thegenerated piece could then be used as a mould for injection moulding orpolymer casting for example. Unfortunately, these methods are limited inresolution, thereby producing a stepwise ladder rather than a slope, andare costly.

Height gradients serve important purposes in microfluidic systems. Forexample, He et al used a slanted feature in a microfluidic mixer toincrease its efficiency by 10%. Microfluidics and Nanofluidics volume19, pages 829-836(2015). Microfluidic channels with trapezoidal crosssection have been applied in centrifugal microfluidics for particleseparation purposes (Scientific Reports volume 3, Article number: 1475(2013), Micromachines (Basel). 2018 April; 9(4): 171. Scientific Reportsvolume 5, Article number: 7717 (2015)). In these cases, the fabricationof such devices has relied on complex, non-scalable manufacturingprotocols such as stereolithography.

Chemical or biomolecule concentration gradients in the microenvironmentplay a significant role in cellular behaviors such as metastasis,embryogenesis, axon guidance, and wound healing (Electrophoresis 2010September; 31(18):3014-27). Since their size is matched to the scale ofthe concentration gradients, microfluidics has become an efficient toolto manipulate fluidic flows and diffusion profiles to createbiomolecular gradients for studying such cellular processes. The methodsfor generating concentration gradients generally exploit branchedconfigurations of rectangular microfluidic channels [RSC Adv., 2017, 7,29966-29984]. Futai et al produced a long-term concentration gradientgenerator by exploiting a height gradient in a microfluidic channelproduced by manipulating the light exposure SU-8 resist to produce aslant in the PDMS mold [Micromachines (Basel). 2019 January; 10(1): 9.]

Lenk et al in Analytical chemistry 90 (22), 13393-13399 demonstrated theuse of assembling a plasma extraction membrane in a slantedconfiguration in front of a microfluidic channel opening to form a wedgelike structure between channel and membrane enabling initiation ofcapillary driven plasma extraction. Hauser et al in Analytical Chemistry2019, 91, 7125-7130 shows a similar device with a pinch-off structurefor a metered volume of extracted plasma and a porous plug forcollecting the plasma. WO 2020/050770 discloses a T-shaped configurationof a metering channel and a bridging element between the meteringchannel and a porous matrix. However, the T-shaped configuration hasproved disadvantageous due to its hematocrit dependency. Thus, thesedevices need improvements to conform with changes in capillarity withinthe device, to control or avoid introduction of air bubbles that maycompromise accuracy or repeated reliable operation for a range ofdifferent blood hematocrit values. Additionally, improvements arenecessary to comply with simple and efficient large scale productionprocesses. For example, WO2011/003689 A2, discloses manufacturingproblems related to slopes for liquid transportation. The formation ofunwanted air bubbles is a general problem in microfluidics. Choi et aladvises a solution with hydrophilic strips to overcome bubble formationwhen a fluid front enters from channel to higher volume compartment. US2009/0152187 discloses a filter chip with plasma separation with anarrowing shape towards an outlet in order to speed up the filtrationprocess. However, there is no disclosure of a metering function or howto balance capillarity in an inlet part of a microfluidic device withplasma separation.

SUMMARY OF INVENTION

An object of the present disclosure is to provide an autonomousmicrofluidic capillary driven device with an inlet and metering sectionfor metering and collecting a sampled body fluid for analysis, with acontrolled capillary transport with a channel system admitting increasedcapillarity.

An object of the present disclosure is to provide an inlet section ofmicrofluidic device with controlled increase capillarity to accesssample such as blood to filtration membrane to support distribution overthe filtration membrane surface to expedite and control the extractionprocess of filtered body fluid such as plasma.

An object of the present disclosure is to introduce a function in amicrofluidic device such that sufficient volume of body fluid isreceived in the device, that relies on simple observations andconvenient user interactions to correct insufficiently received volumes.

An object of the present disclosure is to provide a device that iscapillary driven with a filtration membrane for filtration of bodyfluids that allows for correct separation of a well-defined volume of afiltered body fluid from a remaining fluid plug that consists ofunfiltered body fluid and filtered body fluid.

An object of the present disclosure is to provide a device that iscapillary driven for a filtration of body fluids and with a meteringfunction that relies on air liquid interfaces with controlled air bubbleintroduction to support correct transportation and separation of themetered fluid for collection.

It is also an object of the present disclosure to provide a microfluidicdevice that is able to filter and transport a blood sample, correctlymeter the obtained plasma and separate metered plasma sample, thatreliably operates for all blood hematocrit levels.

It is also an object of the present disclosure to provide a microfluidicdevice that admits a controlled input volume of sample body fluid to bereceived and that correlates with the dead volume of the device and adefined output volume to be collected for analysis.

In general aspects of the present disclosure and in the following, it isreferred to chambers and channels of the system with carefully selectedconfigurations in order to correctly transport, filter, meter andcollect the body fluid. Such configurations will include dimensions ofthe chambers or channels designed to suitably support transportation andseparating and collecting a metered volume. The dimensions can beaddressed in terms of “height”, “width” of the chambers or channels.Other configurations can relate to the materials or other featuresmaking up the chambers or channels and in such contexts terms like“floor” and “roof” will be used. Accordingly, such terms will have anormal meaning for a skilled person. In context of the presentdisclosure, the microfluidic devices are arranged with “a connector”, “afluid connector” or “a connecting piece”. When used, these termsrepresent linking channels or chambers in fluid communication withneighboring parts of device and dimensioned as disclosed to support acapillary transport in the device and may introduce specific functionsto the device.

In general aspects of the present disclosure, the term “capillarity”relates to capillary pressures that exist at liquid-air interfaces,where surface tension, or interface tension exists. Capillarity dependson the dimensions of the device, such as the pore size of a membrane,the type of liquid, such as aqueous or organic, salt content, etc., andthe dimensions and/or surface properties of a flow channel, such ashydrophobic or hydrophilic, including the degree of hydrophobicity orhydrophilicity of surfaces (contact angle). The terms “capillarity” and“capillary pressure” will both be used in various contexts of thepresent disclosure. For example, the term “capillarity” will be used tofunctionally describe features of the device such as channels andchambers. For example, the term “capillary pressure” will for example beused to when describing performing methods of the present disclosure totransport and meter a body fluid by means of the inventive device. A“capillary means” as referred to herein is a porous member that can actas capillary pump and collect the body fluid, for any subsequentanalysis of body fluid constituents.

The term “flow reduction means” has a general meaning in the context ofthe present disclosure that features in channels or chambers of thedevice that temporarily reduce or stop the capillary flow of body fluidfrom an inlet to an outlet of the device. A flow reduction means isexemplified by a capillary stop valve, a dissolvable valve, a part of achannel with altered hydrophilicity, a part of a channel with changeddimensions, and a part a channel with increased flow resistance.

The term “pinch-off means” is used generally to describe parts of thepresent disclosure where a predefined volume of body fluid is separatedfrom the remaining body fluid of the device. In this respect, thepinch-off is established by introducing an air bubble at a region in thedevice with low capillarity, where resistance to the entrance of air isat low point compared to surrounding regions. A “pinch-off means”according to the present disclosure can be located in a pinch-off regiondesigned to induce a low capillary pressure to a transported liquidcolumn that can be used to reduce the flow resistance to introduce andone or more air bubbles from one or more air vents in a pinch-off regionand thereby disconnect a metered liquid volume from the remainingsampled volume with the device.

In general aspects of the present disclosure and in the following, a“capillary means” is a feature acting as a capillary pump and serving tocollect the metered body fluid in the device for subsequent analysis ofone or analytes, optionally in a filtered body fluid. The skilled personwill understand that the capillary means has a controlled porosityadapted to other parts of the device, as further explained inWO2015/044454. In general aspects of the present disclosure and in thefollowing, the term “body fluid” can relate to blood and the filteredbody fluid is plasma. Other body fluids for transportation, metering andcollection would also be conceivable to perform with device.

In a first aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport,wherein the device comprises: an inlet section for receiving a sample ofbody fluid, the inlet section comprising an inlet port and a channelsystem configured to transport the sample of body fluid; a filtrationmembrane configured to separate plasma from blood; a metering section,configured to meter a predefined volume of the received body fluid anddisconnect it from remaining fluid in the device; and an outlet sectionconfigured to receive and collect the metered volume of body fluid fromthe metering section, the outlet section comprising a capillary meansfor collection of the metered volume, wherein the channel systemcomprises consecutively in the flow direction a first channel arrangedin fluid communication with the inlet port, a second channel and a thirdchannel, wherein the inlet section and the channel system are configuredto transport the sample of body fluid to, and to distribute it acrossthe filtration membrane with a stepwise or gradually increasingcapillarity from the inlet section to the filtration membrane; themetering section comprises an extraction chamber configured to receivean extracted body fluid from the filtration membrane and arranged influid communication with a metering channel; and the metering sectioncomprises a pinch-off means configured to separate the metered volume ofbody fluid, wherein the pinch-off means comprises at least one air ventarranged in a part of the extraction chamber with the maximum height.

By means of the stepwise or gradual increase in capillarity, it isensured that the sample of body fluid is transported from the inletsection to the filtration membrane without pinning to guaranteecontinuous operation of the device. Additionally, the stepwise orgradual increase in capillarity enables distribution across the membranesuch that filtration occurs substantially evenly throughout themembrane. By means of the air vent, an effective separation of themetered volume from the remaining volume of body fluid is achieved.

In one embodiment, the stepwise or gradual increase in capillarity ofthe channel system is established by successively, from the inlet portto the filtration membrane, decreasing the height of the channels and/orsuccessively increasing the hydrophilicity of the channels.

In one embodiment, a floor of the third channel is defined by a flatupper surface of the filtration membrane. Thus, the third channelextends parallel to the filtration membrane forming a filtrationchamber.

In one embodiment, a height ratio of the first channel to the secondchannel is at least 1.1:1, preferably at least 2:1, and wherein a heightratio of the second channel to the third channel is at least 1.1:1,preferably at least 2:1, preferably the height of the first channel is500-2000 μm; the height of the second channel is 100-600 μm; and theheight of the third channel is 25-200 μm.

In one embodiment, the second channel comprises a capillary stop valveand a means for visual filling inspection, such as an inspection window,both located adjacent to the first channel outlet. By means of thecapillary stop valve, flow of body fluid through the channel system maybe interrupted until supply of body fluid is removed from the inletport, whereby the capillary stop valve bursts through increase inLaplace pressure on the droplet forming at the inlet port whichovercomes the threshold pressure of the capillary stop valve. This maybe used to meter the volume of the body fluid before it flows into thesecond channel. The user can check the level of filling in the means forvisual inspection to ensure that a sufficient amount has been supplied.

In one embodiment, the capillary stop valve is selected from at leastone of a part of the second channel with altered hydrophilicity and/or apart of the second channel with changed dimensions. The hydrophilicityand/or dimension of the second channel may be configured to achieve thedesired threshold or burst pressure of the capillary stop valve.Preferably, the capillary stop valve is formed by an abrupt increase inheight in the second channel.

In one embodiment, the pinch-off means comprises a pinch-off region,arranged in fluid communication with one or more air vents locatedbefore the entrance to the metering channel, wherein the pinch-offregion comprises a height reducing element with a height lower than themaximum height of the extraction chamber. Preferably, the heightreducing element has a through-hole to prevent from liquid pinning inthe extraction chamber.

In one embodiment, the extraction chamber comprises a part withgradually increasing height, a part with the height reducing element anda part with a maximum height arranged in fluid communication with themetering channel.

In one embodiment, a roof of the extraction chamber is defined by a flatlower surface of the filtration membrane and a floor of the extractionchamber extends at an acute angle from a contact with the filtrationmembrane towards the metering channel. Preferably, the extractionchamber is generally wedge-shaped with a gradually increasing heightfrom a contact point with the filtration membrane towards the meteringchannel and, wherein the maximum height of the extraction chamberexceeds the height of the metering channel. By means of the acute anglebetween the filtration membrane and the floor of the extraction chamber,it is possible to achieve a wedge-shaped extraction chamber whichdiverges towards the metering channel, thus enabling gradual filling ofthe space between the diverging surfaces, essentially forming acapillary pump. At the same time, it is possible to maintain asubstantially flat, horizontal orientation of the filtration membrane,which facilitates integrating the filtration membrane in a chamberconstruction to protect a blood sample from evaporation andcontamination during plasma extraction.

In one embodiment, the first channel has a volume correlated to the deadvolume and the metered volume (the output volume) of the device.Preferably, the volume of the first channel is sufficient to prevent afront meniscus of a body fluid volume other than the metered volume fromreaching the capillary means of the outlet section. The dead volume isthe sum of all volumes that are not metered and collected in thecapillary means at the outlet. In other words, the dead volume is theresidual volume in the system which is distributed across the filtrationchamber, the plasma extraction (filtration) membrane and the plasmaextraction chamber. The plasma output (metered) volume is the volumethat is separated from the dead volume, e.g., by a pinch-off effect. Asthe input volume applied to the inlet port by the user of the devicewill vary and the metered output volume is constant and predetermined bythe device, the dead volume will also be variable within an acceptablerange. Accordingly, the volume of the first channel is correlated to thedead volume and the output metered volume. By selecting the volume ofthe first channel in this way, it is ensured that only the necessaryamount of blood required for the plasma sampling is admitted into thefirst channel.

In one embodiment, the metering channel has an outlet part with adimensional change configured to cause a fluid front meniscus of theseparated metered volume of body fluid, when transported to the outletsection, to assume a shape which substantially conforms to the surfacegeometry of the capillary means. By means of the dimensional change inthe outlet part of the metering channel, the shape of the fluid frontmeniscus can be adapted to the geometry of the capillary means such thatthe shapes at the interface match each other. Thereby, the impact of theseparated metered volume of body fluid with the capillary means can becontrolled to prevent bubble formation between the two medias.

In one embodiment, the dimensional change comprises a reduction in widthand/or height of the metering channel. By reducing the width and/orheight, it is possible to induce forming of a substantially straight orplanar meniscus, overcoming any effects of surface roughness ordimensional variances of the metering channel.

In one embodiment, a distal end of the outlet part of the meteringchannel adjacent the capillary means has a constant width which issmaller than the width of the metering channel. Preferably, the outletpart of the metering channel has a first part with a gradual reductionin width and second part with a constant width which is smaller than thewidth of the metering channel. The reduction in width causes the fluidmeniscus to go from a convex shape to a substantially planar shape whichmatches the geometry of the capillary means.

In one embodiment, the surface geometry of the capillary means at aninterface surface with the fluid front meniscus is curved orsubstantially planar.

In one embodiment, the outlet section comprises a hydrophilic porousbridge element with an average pore size smaller than the smallestdimension of the metering channel, and wherein the bridge element isarranged in fluid communication with the outlet part of the meteringchannel and with the capillary means. By providing a capillary means intwo components, it is possible to introduce an increasing capillarity toensure transport of the separated metered volume of body fluid from themetering channel to the paper substrate for collection.

Additionally, the first aspect of the present disclosure relates to amethod for sampling, transporting and collecting a metered volume ofbody fluid for analysis by means of capillary transport in amicrofluidic device, the method comprising the steps of: applying asupply of body fluid to an inlet port of the device; filling a channelsystem arranged in fluid communication with the inlet port, wherein thechannel system comprises consecutively in the flow direction a firstchannel arranged in fluid communication with the inlet port, a secondchannel and a third channel; transporting a sample of body fluid with astepwise or gradually increasing capillarity to a filtration membraneconfigured to separate plasma from blood; distributing the sample ofbody fluid across the filtration membrane; receiving filtered body fluidin a metering section comprising an extraction chamber, and a meteringchannel in fluid communication with the extraction chamber; transportingthe filtered body fluid in the metering channel to an outlet sectioncomprising a capillary means for collection of the filtered body fluid;disconnecting a metered volume of filtered body fluid by introducing atleast one air bubble in a part of metering section inducing the lowestcapillary pressure; and collecting the metered volume of filtered bodyfluid in the capillary means.

In one embodiment, the method is performed with a device according tothe first aspect with a sample of blood to meter and collect bloodplasma.

In a second aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport,wherein the device comprises: an inlet section for receiving a sample ofbody fluid, the inlet section comprising an inlet port and a channelsystem; a filtration membrane configured to separate plasma from blood,wherein the inlet section and the channel system are configured totransport the sample of body fluid to, and to distribute it across thefiltration membrane with a stepwise or gradually increasing capillarityfrom the inlet section to the filtration membrane; a metering function,configured to meter a predefined volume of the received body fluid; andat least one porous medium for receiving the transported sample of bodyfluid.

By means of the stepwise or gradual increase in capillarity, it isensured that the sample of body fluid is transported from the inletsection to the filtration membrane without pinning to guaranteecontinuous operation of the device. Additionally, the stepwise orgradual increase in capillarity enables distribution across the membranesuch that filtration occurs substantially evenly throughout themembrane.

In one embodiment, the channel system comprises at least two channels,including a first channel arranged in fluid communication with the inletport and with a second channel having a higher capillarity than thefirst channel. In one embodiment, a height ratio of the first channel tothe second channel is at least 1.1:1, preferably at least 2:1, With atleast two channels, the increase in capillarity can be achieved in atleast two steps, for instance through a height reduction.

In one embodiment, the channel system comprises at least one of a flowreduction means and a means for visual filling inspection, such as aninspection window. Preferably, the means for filling inspection isprovided in the second channel adjacent to the first channel. The flowreduction means and filling inspection means enable pre-metering byinterrupting the flow of the sample such that the operator may stopapplication of body fluid to the device when a sufficient amount hasbeen added, i.e., the channel system has been filled.

In one embodiment, the flow reduction means is selected from at leastone of: a part of the second channel with altered hydrophilicity; a partof the second channel with changed dimensions; and a part of the secondchannel with increased flow resistance, preferably the flow reductionmeans is provided adjacent to the means for visual inspection.Preferably, the flow reduction means is a dissolvable valve or acapillary stop valve, preferably the capillary stop valve comprises anabrupt increase in the second channel height.

In one embodiment, the porous medium is configured to absorb and collecta received volume, preferably the porous flow medium is a lateral flowmedium or a filter paper.

In one embodiment, the metering function comprises a metering sectionwith an extraction chamber configured to receive an extracted body fluidfrom the filtration membrane and arranged in fluid communication with ametering channel, and wherein the device further comprises an outletsection configured to receive and collect the metered volume of bodyfluid from the metering channel, the outlet section comprising acapillary means for collection of the metered volume.

In one embodiment, the channel system comprises a first channel having afirst capillarity and arranged in fluid communication with the inletport and with a third channel having a second capillarity, the secondcapillarity being higher than the first capillarity, and wherein thethird channel comprises a roof, optionally a vent, and is configured tohomogenously distribute the sample of body fluid arriving from the firstchannel across the filtration membrane. Preferably, the third channelcomprises a floor defined by a flat upper surface of the filtrationmembrane.

In one embodiment, the stepwise or gradual increase in capillarity ofthe channel system is established by successively, from the inlet portto the filtration membrane, decreasing the height of the channels and/orincreasing the hydrophilicity of the channels. Preferably, the stepwiseincrease in capillarity of the channel system from the inlet port to thefiltration membrane is established over at least two steps.

In one embodiment, the first channel has a volume correlated to the deadvolume and the metered volume of the device, preferably the volume ofthe first channel is sufficient to prevent a front meniscus of a bodyfluid volume other than the metered volume from reaching the capillarymeans of the outlet section. The dead volume is the sum of all volumesthat are not metered and collected in the capillary means at the outlet.In other words, the dead volume is the residual volume in the systemwhich is distributed across the filtration chamber, the plasmaextraction (filtration) membrane and the plasma extraction chamber. Theplasma output (metered) volume is the volume that is separated from thedead volume, e.g., by a pinch-off effect. As the input volume applied tothe inlet port by the user of the device will vary and the meteredoutput volume is constant and predetermined by the device, the deadvolume will also be variable within an acceptable range. Accordingly,the volume of the first channel is correlated to the dead volume and theoutput metered volume. By selecting the volume of the first channel inthis way, it is ensured that only the necessary amount of blood requiredfor the plasma sampling is admitted into the first channel.

In one embodiment, the device further comprises a second channelarranged between and in fluid communication with the first channel andthe third channel. The second channel provides an additional step in thechannel system to achieve the stepwise or gradual increase incapillarity. Preferably, the height ratio of the second channel to thethird channel is at least 1.1:1, preferably at least 2:1.

In one embodiment, the extraction chamber is generally wedge-shaped witha gradually increasing height from a contact with the filtrationmembrane towards the metering channel, and wherein the maximum height ofthe extraction chamber is higher than the height of the meteringchannel. The wedge shape enables gradual filling of the extractionchamber.

In one embodiment, the device further comprises a pinch-off meansconfigured to separate the metered volume of body fluid, wherein thepinch off means comprises at least one air vent arranged in a part ofthe extraction chamber with the maximum height. By means of the airvent, an effective separation of the metered volume from the remainingvolume of body fluid is achieved.

In one embodiment, the pinch-off means comprises a pinch-off region influid communication with the at least one air vent, arranged adjacent anentrance to the metering channel, wherein the pinch-off region comprisesa height reducing element with a height lower than the maximum height ofthe extraction chamber. Preferably, the extraction chamber comprises apart with gradually increasing height, a part with a height reducingelement and a part with a maximum extraction chamber height in fluidcommunication with the metering channel. The height reducing elementcreates an increase in capillarity at the egress of the extractionchamber, thus ensuring continued transport and filtration of the bodyfluid through the filtration membrane.

In one embodiment, the height reducing element comprises a through-hole,to prevent liquid pinning.

Additionally, the second aspect of the present disclosure relates to amethod of sampling, metering and collecting a body fluid sample foranalysis by means of a microfluidic device as embodied in this secondaspect. The method comprises applying a sample volume to an inlet portof the device and transporting the sample volume to a porous filtrationmembrane through a channel system admitting a successive increase incapillary pressure, preferably a stepwise increase of capillarypressure. The method further comprises admitting a still increasedcapillary pressure from a porous filtration membrane to separatecellular material and to extract remaining body fluid; receiving afiltered body fluid from the filtration membrane in an extractionchamber inducing gradually lower capillary pressure; filling a meteringchannel with the filtered body fluid by means of an increased capillarypressure; and disconnecting the fluid communication between theextraction chamber and the metering channel by introducing an air bubbleat a point predetermined to subject the body fluid to the lowestcapillary pressure; and collecting the metered body fluid in a capillarymeans comprised in an outlet section. Preferably, the fluidcommunication between the extraction chamber and the metering channel isdisconnected when the metered body fluid contacts the capillary means.

In embodiments of the method, a volume of the body fluid is manuallyapplied the inlet port; from the inlet port the body fluid is admittedto fill a first channel, whereupon once the first channel is filled, aflow reduction means temporarily stops or reduces the body fluidtransport. After safeguarding that the device is correctly filled,excess body fluid is removed from the inlet port so further transport isadmitted to the separating, metering and collection procedures.

In a third aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport,wherein the device comprises: an inlet section, for receiving a sampleof body fluid, the inlet section comprising an inlet port; a meteringsection configured to receive body fluid from the inlet section andcomprising a metering channel, wherein the metering section is arrangedto separate a metered volume of body fluid filled in the meteringchannel; and an outlet section configured to receive and transport theseparated metered volume of body fluid for collection in a capillarymeans having a predetermined surface geometry, wherein the meteringchannel has an outlet part with a dimensional change configured to causea fluid front meniscus of the separated metered volume of body fluid,when transported to the outlet section, to assume a shape whichsubstantially conforms to the surface geometry of the capillary means.

By means of the dimensional change in the outlet part of the meteringchannel, the shape of the fluid front meniscus can be adapted to thegeometry of the capillary means such that the shapes at the interfacematch each other. Thereby, the impact of the separated metered volume ofbody fluid with the capillary means can be controlled to prevent bubbleformation between the two medias.

In one embodiment, the dimensional change comprises a reduction in widthand/or height of the metering channel. By reducing the width and/orheight, it is possible to induce forming of a substantially straight orplanar meniscus, overcoming any effects of surface roughness ordimensional variances of the metering channel.

In one embodiment, a distal end of the outlet part of the meteringchannel adjacent the capillary means has a constant width which issmaller than the width of the metering channel. Preferably, the outletpart of the metering channel has a first part with a gradual reductionin width and second part with a constant width which is smaller than thewidth of the metering channel. The reduction in width causes the fluidmeniscus to go from a convex shape to a substantially planar shape whichmatches the geometry of the capillary means.

In one embodiment, the surface geometry of the capillary means at aninterface surface with the fluid front meniscus is curved orsubstantially planar.

In one embodiment, the capillary means comprises a bridge elementarranged in fluid communication with the outlet part of the meteringchannel and a paper substrate connected to the bridge element.Preferably, the bridge element is a hydrophilic porous element with anaverage pore size smaller than the smallest dimension of the meteringchannel. By providing a capillary means in two components, it ispossible to introduce an increasing capillarity to ensure transport ofthe separated metered volume of body fluid from the metering channel tothe paper substrate for collection.

In one embodiment, the bridge element, is made from a material selectedfrom at least one of micro paper pulp, micro fibrillated cellulose, anopen cell hydrophilic polymer or a highly compressible glass fiber web.

In one embodiment, the surface geometry of the bridge element at aninterface surface with the fluid front meniscus is curved orsubstantially planar.

In one embodiment, the device further comprises a filtration membraneconfigured to separate selected cells from the body fluid, wherein theinlet section is configured to transport the sample of body fluid to,and to distribute it across the filtration membrane and wherein themetering section comprises an extraction chamber configured to receivebody fluid from the filtration membrane and to transport the receivedbody fluid to the metering channel. By means of the filtration membrane,it is possible to separate e.g., plasma from whole blood for collectionin the capillary means.

In one embodiment, the device further comprises a pinch-off meansconfigured to separate the metered volume of body fluid, wherein thepinch-off means comprises at least one air vent arranged in a part ofthe extraction chamber with a maximum height. By means of the air vent,an effective separation of the metered volume from the remaining volumeof body fluid is achieved.

In one embodiment, the pinch-off means comprises a pinch-off region influid communication with the at least one air vent, the pinch-off regionbeing arranged in the part of the extraction chamber with the maximumheight and surrounded by areas with lower height. Preferably, at leastone part of the extraction chamber surrounding the pinch-off region hasa height lower than the height of the metering channel. The surroundingareas of lower height lead to a reduction of the capillary pressure inthe pinch-off region, thus promoting introduction of an air bubble.

In one embodiment, the metering section comprises a fluid connectorextending between the extraction chamber and the metering channel, andan air vent. The air vent may be arranged adjacent to, or at theposition where the fluid connector meets the metering channel.Preferably, the air vent is arranged at the entrance of the meteringchannel and is configured as an orifice to ambient air with across-sectional area equal to or greater than the size of thecross-sectional area of the metering channel. The air vent is thusplaced in a location of the device with low capillary pressure, optimalfor introducing an air bubble downstream of the extraction chamber andupstream of the metering channel to separate the metered volume of bodyfluid.

In one embodiment, the fluid connector has a different dimension thanthe metering channel, the dimension being selected from one or more ofheight, width and length. Preferably, the fluid connector has agradually increasing height towards the entrance of the meteringchannel. Thereby, the fluid/air interface is increased to facilitateintroduction of an air bubble.

In one embodiment, the maximum height of the extraction chamber is lowerthan the height of the metering channel.

Additionally, the third aspect of the present disclosure relates to amethod for sampling, transporting and collecting a metered volume ofbody fluid for analysis by means of capillary transport from an inlet toa capillary means of a microfluidic device, the method comprising thesteps of: applying a sample of body fluid to an inlet port of the deviceand transporting the body fluid, optionally through a filtrationmembrane, to a metering channel; admitting the metering channel totransport the sample of body fluid to an outlet section comprising acapillary means having a predetermined surface geometry; receiving themetered fluid in the capillary means and separating a metered volume ofbody fluid from the remaining sample volume by introducing at least oneair bubble at a point of the device upstream of the metering channelexhibiting low capillary pressure; and collecting the metered volume ofbody fluid in the capillary means, wherein an outlet part of themetering channel comprises a dimensional change which causes a fluidfront meniscus of the separated metered volume of body fluid, whentransported to the outlet section, to assume a shape which substantiallyconforms to the surface geometry of the capillary means.

In a fourth aspect of the present disclosure, there is provided a methodof manufacturing an outlet section of a microfluidic device configuredto sample, meter and collect a metered volume of body fluid for analysisby means of capillary transport; the method comprising: providing amicrofluidic device having an outlet section in fluid communication witha metering section comprising a metering channel configured to receivebody fluid from an inlet section with an inlet port, wherein the outletsection comprises a bridge cavity between an outlet part of the meteringchannel and an outlet orifice of the device; providing a hydrophilicporous bridge element arranged to conform to the shape of the bridgecavity; inserting the bridge element into the bridge cavity, such thatthe bridge element substantially fills the bridge cavity and the outletorifice; and attaching a capillary means to the outlet section, therebyestablishing contact between the capillary means and the bridge element.

By inserting a conformable hydrophilic porous bridge element into thebridge cavity in such a way that the bridge cavity is substantiallyfilled, the need for high precision cutting and placement of a porouselement into the outlet is reduced or eliminated. Instead, the methodaccording to the fourth aspect enables application of the solution inautomatized high throughput mass manufacturing.

In one embodiment, inserting causes the bridge element to protrude intothe metering channel. Preferably, inserting causes a surface of the partof the bridge element which protrudes into the metering channel toassume a shape which substantially conforms to a fluid front meniscus ofa metered volume of body fluid in the metering channel. Thus, the impactof the separated metered volume of body fluid with the bridge elementcan be controlled to prevent bubble formation between the two medias.

In one embodiment, the bridge element is made of a compressible porousmaterial and has a volume which is larger than a volume of the bridgecavity, and wherein inserting comprises compressing the bridge elementinto the bridge cavity. With a compressible material, the bridge elementis simply inserted by compressing it into the bridge cavity and ensuresthat no gaps are formed between the bridge cavity and the bridgeelement.

In one embodiment, the bridge element is made of a dispensable porousmaterial, and wherein inserting comprises dispensing the porous materialinto the bridge cavity such that it protrudes outside the outlet orificeand allowing the porous material to set to form the bridge element. Witha dispensable material, the bridge element is simply dispensed into thebridge cavity and ensures that no gaps are formed between the bridgecavity and the bridge element. In this context, dispensable materialencompasses any suitable material e.g., in liquid form which may bedispensed through a nozzle or similar into the bridge cavity andsubsequently cure or set into solid form.

In one embodiment, the capillary means is configured to exert a highercapillary pressure on the body fluid than the bridge element, andwherein the bridge element has an average pore size smaller than thesmallest dimension of the metering channel. This ensures that the sampleof body fluid is transported from the metering channel through thebridge element to the capillary means.

In one embodiment, the bridge element is made from a material selectedfrom at least one of micro paper pulp, micro fibrillated cellulose, anopen cell hydrophilic polymer or a highly compressible glass fiber web.

Additionally, the fourth aspect relates to a microfluidic deviceconfigured to sample, meter and collect a metered volume of body fluidfor analysis by means of capillary transport, the device comprising: aninlet section, for receiving the body fluid sample, the inlet sectioncomprising an inlet port; a metering section configured to receive bodyfluid from the inlet section and comprising a metering channel, whereinthe metering section is arranged to separate a metered volume of bodyfluid filled in the metering channel; and an outlet section comprising abridge cavity between an outlet part of the metering channel and anoutlet orifice of the device, a hydrophilic porous bridge elementarranged to conform to the shape of the bridge cavity and inserted inthe bridge cavity such that the bridge element substantially fills thebridge cavity and the outlet orifice, and a capillary means attached tothe outlet section in contact with the bridge element.

In one embodiment, the device further comprises a filtration membraneconfigured to separate selected cells from the body fluid, wherein theinlet section is configured to transport the sample of body fluid to,and to distribute it across the filtration membrane and the meteringsection comprises an extraction chamber configured to receive body fluidfrom the filtration membrane and to transport the received body fluid tothe metering channel. By means of the filtration membrane, it ispossible to separate e.g., plasma from whole blood for collection in thecapillary means.

In one embodiment, the metering section comprises a fluid connectorextending between the extraction chamber and the metering channel, andan air vent. The air vent may be arranged adjacent to, or at theposition where the fluid connector meets the metering channel. The airvent is thus placed in a location of the device with low capillarypressure, optimal for introducing an air bubble downstream of theextraction chamber and upstream of the metering channel to separate themetered volume of body fluid. Preferably, the fluid connector has adifferent dimension than the metering channel, the dimension beingselected from one or more of height, width and length.

In one embodiment, the outlet part of the metering channel is configuredto cause a fluid front meniscus of the separated metered volume of bodyfluid, when transported to the outlet section, to assume a shape whichsubstantially conforms to the surface geometry of the capillary means.Preferably, the surface of the bridge element facing the meteringchannel is curved or substantially planar. Thus, the impact of theseparated metered volume of body fluid with the bridge element can becontrolled to prevent bubble formation between the two medias.

In one embodiment, the device further comprises a pinch-off meansconfigured to separate the metered volume of body fluid, wherein thepinch-off means comprises at least one air vent arranged in a part ofthe extraction chamber with a maximum height. By means of the air vent,an effective separation of the metered volume from the remaining volumeof body fluid is achieved.

In one embodiment, the pinch-off means comprises a pinch-off region influid communication with the at least one air vent, the pinch-off regionbeing arranged in the part of the extraction chamber with the maximumheight and surrounded by areas with lower height. Preferably, at leastone part of the extraction chamber surrounding the pinch-off region hasa height lower than the height of the metering channel. The surroundingareas of lower height lead to a reduction of the capillary pressure inthe pinch-off region, thus promoting introduction of an air bubble.

In one embodiment, the maximum height of the extraction chamber is lowerthan the height of the metering channel.

In one embodiment, the extraction chamber is substantially wedge-shaped,wherein a roof of the extraction chamber is defined by flat lowersurface of the filtration membrane, and wherein a hydrophilic floor ofthe extraction chamber extends at an acute angle from a contact with thefiltration membrane towards the metering channel. By means of the acuteangle between the filtration membrane and the floor of the extractionchamber, it is possible to achieve a wedge-shaped extraction chamberwhich diverges towards the metering channel, thus enabling gradualfilling of the space between the diverging surfaces, essentially forminga capillary pump. At the same time, it is possible to maintain asubstantially flat, horizontal orientation of the filtration membrane,which facilitates integrating the filtration membrane in a chamberconstruction to protect a blood sample from evaporation andcontamination during plasma extraction. Preferably, the hydrophilicfloor is the floor of a fluid connector extending between the extractionchamber and the metering channel.

In one embodiment, the fluid connector has a maximum height and aminimum height which is smaller than the maximum height of theextraction chamber.

In a fifth aspect of the present disclosure, there is provided amultilayer microfluidic device configured to sample, meter and collect ametered volume of body fluid for analysis by means of capillarytransport, wherein the device comprises: an inlet section for receivinga body fluid sample, the inlet section comprising an inlet port and isconfigured to transport and access the sample to a flat, laterallyextending filtration membrane; a metering section, comprising anextraction chamber and a metering channel, the extraction chamber beingconfigured to receive an extracted body fluid from the filtrationmembrane and arranged in fluid communication with the metering channel;and an outlet section configured to receive and collect a metered volumeof body fluid from the metering channel, the outlet section comprising acapillary means for collection of the metered volume of body fluidwherein a roof of the extraction chamber is defined by a flat lowersurface of the filtration membrane, and a floor of the extractionchamber is continuous with a floor of the metering channel and extendsat an acute angle from the lower surface of the filtration membrane, andwherein the floor of the extraction chamber is inclined with respect tothe floor of the metering channel to create a slope.

By means of the inclined floor of the extraction chamber it is possibleto achieve a wedge-shaped extraction chamber which diverges towards themetering channel, thus enabling gradual filling of the space between thediverging surfaces, essentially forming a capillary pump. At the sametime, it is possible to maintain a substantially flat, horizontalorientation of the filtration membrane, which facilitates integratingthe filtration membrane in a chamber construction to protect a bloodsample from evaporation and contamination during plasma extraction.

In one embodiment, the device comprises from the bottom to the top: abottom layer; a hydrophilic floor layer forming the floor of theextraction chamber and the metering channel; and a support structure forthe floor layer, wherein the support structure is arranged between thebottom layer and the floor layer such that a first part of the floorlayer is supported on the support structure to contact the filtrationmembrane, and wherein a second part of the floor layer is supported onthe bottom layer to form the acute angle between the filtration membraneand the floor layer in order to obtain an extraction chamber with aheight gradually increasing towards the metering channel. By means ofthe layer construction, assembly of the device is facilitated to enablescalable mass manufacturing.

In one embodiment, the device comprises at least five layers selectedfrom: the bottom layer; the support structure; the floor layer; achannel structure layer configured to accommodate the metering section;and a cover layer providing a flat roof surface for the meteringchannel.

In one embodiment, the floor layer comprises a slot delimiting a tongueportion which forms the floor of the extraction chamber, and wherein afree end of the tongue portion is supported on the support structure.Preferably, the slot is substantially C-shaped and the tongue portion issubstantially circular or substantially square. By means of the slot, adesired shape of the tongue portion to form the floor of the extractionchamber can easily be cut, e.g., be adapted to the shape of thefiltration membrane.

In one embodiment, the floor layer comprises an opening forming anoutlet port of the outlet section.

In one embodiment, the bottom layer comprises a first openingsubstantially corresponding to the size of the extraction chamber and asecond opening arranged to accommodate the capillary means.

In one embodiment, the channel structure layer comprises an openingarranged to accommodate the support structure, the floor of theextraction chamber and an outlet port of the outlet section, preferablysaid channel structure layer further comprises a slot forming side wallsof the metering channel.

In one embodiment, the cover layer comprises an opening substantiallycorresponding to the size of the extraction chamber, and wherein thelower surface of the filtration membrane is positioned thereon.

The openings in the different layers accommodate the differentstructures forming the microfluidic device, enabling the multilayerconstruction.

In one embodiment, the cover layer has a first side facing the channelstructure layer with a hydrophilic surface and a second, opposite sidewith an adhesive surface. The hydrophilic surface thus forms the roof ofthe metering channel, and the adhesive surface enables assembly ofadditional layers on top of the cover layer.

In one embodiment, the device further comprises at least one additionallayer attached to the second side of the cover layer for assembling theinlet section and a device housing.

Additionally, the fifth aspect of the present disclosure relates to amethod of manufacturing a microfluidic device by lamination of foillayers, comprising the steps of: providing a substrate as a bottom layerof the device; assembling a support structure on the bottom layer;providing a floor layer with a hydrophilic upper surface and assemblingthe floor layer on the bottom layer such that a first part of the floorlayer is supported on the support structure and a second part of thefloor layer is supported on the bottom layer, wherein the first part ofthe floor layer is inclined with respect to the second part to create aslope; providing a channel structure layer configured to accommodate ametering section and assembling the channel structure layer on thechannel floor layer; providing a cover layer and assembling the coverlayer on the channel structure layer; and assembling a filtrationmembrane in a horizontal position to rest on the cover layer, therebycreating an extraction chamber with the first part of the floor layer asa floor.

By means of the manufacturing method, scalable mass production of amulti-layered microfluidic device with a wedge-shaped extraction chamberis enabled.

In one embodiment, the method further comprises forming a slot in thefloor layer to delimit a tongue portion which forms the first part, andassembling the floor layer on the bottom layer such that a free end ofthe tongue portion is supported on the support structure.

In one embodiment, the floor layer comprises an opening forming anoutlet port of the outlet section.

In one embodiment, the bottom layer comprises a first openingsubstantially corresponding to the size of the extraction chamber and asecond opening arranged to accommodate the capillary means.

In one embodiment, the channel structure layer comprises an openingarranged to accommodate the support structure, the floor of theextraction chamber and an outlet port of the outset section.

In one embodiment, the cover layer has a first side facing the channelstructure layer with a hydrophilic surface and a second, opposite sidewith an adhesive surface.

In one embodiment, the method further comprises assembling at least oneadditional layer on the cover layer and subsequently assembling an inletsection and a housing on the at least one additional layer.

In a sixth aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport,wherein the device comprises: an inlet section, for receiving a bodyfluid sample, the inlet section comprising an inlet port arranged toreceive a supply of body fluid; a metering function configured toreceive body fluid from the inlet section and comprising a firstchannel; and a sequent section configured to receive the body fluid fromthe metering function and comprising a second channel, wherein the firstchannel comprises a capillary stop valve configured to interrupt orreduce flow of the body fluid therethrough, and a means for visualinspection arranged adjacent to the capillary stop valve, wherein ageometry and/or dimension of the inlet port is configured such that whenthe supply of body fluid to the inlet port is removed, the Laplacepressure of a body fluid meniscus at the inlet port is higher than athreshold pressure of the capillary stop valve.

By configuring the geometry and/or dimension of the inlet port, adesired curvature of the meniscus of the body fluid, which sticks to theinlet port when the supply of body fluid is removed, can be achieved. Inone embodiment, the body fluid is blood from a finger prick which isapplied to the inlet port. The curvature of the meniscus in turndetermines the Laplace pressure induced by the surface tension on theliquid. By selecting a geometry and/or dimension of the inlet port insuch a way that the Laplace pressure on the body fluid at the inlet portis higher than the threshold pressure of the capillary stop valve, thiswill lead to bursting of the capillary stop valve when the supply ofbody fluid (e.g. a blood droplet on the finger) is removed to allow thebody fluid to flow from the first channel to the second channel. Thismay be used to meter the volume of the body fluid before it flows intothe second channel. The user can check the level of filling in the meansfor visual inspection to ensure that a sufficient amount has beensupplied.

In one embodiment, the capillary stop valve is selected from at leastone of a part of the first channel with altered hydrophilicity and/or apart of the first channel with changed dimensions. The hydrophilicityand/or dimension of the first channel may be configured to achieve thedesired threshold or burst pressure of the capillary stop valve.Preferably, the capillary stop valve is formed by an abrupt increase inheight in the first channel.

In one embodiment, the sequent section comprises at least one porousmedium for receiving or collecting body fluid from the first channel.Thus, a sample of body fluid may be collected in a simple and efficientmanner.

In one embodiment, a height ratio of the first channel to the secondchannel is at least 1.1:1, preferably at least 2:1. The difference inheight ensures continued capillary transport from the first channel tothe second channel.

In one embodiment, a surface surrounding the inlet port is hydrophobic.The hydrophobic surface aids in forming a droplet of the body fluidwhich sticks to the inlet port, thereby increasing the Laplace pressure.

In one embodiment, the metering function is a pre-metering function ofblood and the first channel is a pre-metering channel arranged in fluidcommunication with a filtration membrane and an extraction chamberconfigured to receive body fluid from the filtration membrane and totransport it to and fill a plasma metering channel. By means of thefiltration membrane, the extraction chamber and the plasma meteringchannel, the device is further configured to autonomously separate,meter and collect plasma from blood, preferably in a capillary meansarranged in fluid communication with the plasma metering channel.

In one embodiment, the device further comprises a pinch-off meansconfigured to separate the metered volume of body fluid, wherein thepinch-off means comprises at least one air vent arranged in a part ofthe extraction chamber with a maximum height. By means of the air vent,an effective separation of the metered volume from the remaining volumeof body fluid is achieved.

In one embodiment, the pinch-off means comprises a pinch-off region influid communication with the at least one air vent and arranged adjacentthe part of the extraction chamber with the maximum height andsurrounded by areas with lower height. Preferably, at least one areasurrounding the pinch-off region has a height lower than a height of theplasma metering channel. The surrounding areas of lower height lead to areduction of the capillary pressure in the pinch-off region, thuspromoting introduction of an air bubble.

In one embodiment, the device further comprises a fluid connectorextending between the extraction chamber and the plasma meteringchannel, and an air vent. The air vent may be arranged adjacent to, orat the position where the fluid connector meets the plasma meteringchannel. Preferably, the air vent is arranged at the entrance of theplasma metering channel and is configured as an orifice to ambient airwith a cross-sectional area equal to or greater than the size of thecross-sectional area of the plasma metering channel. The air vent isthus placed in a location of the device with low capillary pressure,optimal for introducing an air bubble downstream of the extractionchamber and upstream of the plasma metering channel to separate themetered volume of body fluid.

In one embodiment, the fluid connector has a different dimension thanthe plasma metering channel, the dimension being selected from one ormore of height, width and length.

In one embodiment, a maximum height of the extraction chamber is lowerthan the height of the plasma metering channel.

In one embodiment, the extraction chamber is substantially wedge-shapedwith a gradually increasing height, wherein a roof of the extractionchamber is defined by a flat lower surface of the filtration membrane,and wherein a hydrophilic floor of the extraction chamber extends at anacute angle from a contact with the filtration membrane towards theplasma metering channel. By means of the acute angle between thefiltration membrane and the floor of the extraction chamber, it ispossible to achieve a wedge-shaped extraction chamber which divergestowards the plasma metering channel, thus enabling gradual filling ofthe space between the diverging surfaces, essentially forming acapillary pump. At the same time, it is possible to maintain asubstantially flat, horizontal orientation of the filtration membrane,which facilitates integrating the filtration membrane in a chamberconstruction to protect a blood sample from evaporation andcontamination during plasma extraction.

Additionally, the sixth aspect of the present disclosure relates to amethod for sampling, transporting and collecting a metered volume ofbody fluid for analysis by means of capillary transport in amicrofluidic device, the method comprising the steps of: manuallyapplying a supply of body fluid to an inlet port of the device; fillinga first channel arranged in fluid communication with inlet port withbody fluid by means of capillary pressure, wherein the first channelcomprises a capillary stop valve configured to interrupt or reduce flowof the body fluid therethrough; visually inspecting the first channelfor correct filling; removing the supply of body fluid to the inletport, wherein a geometry and/or dimension of the inlet port isconfigured such that when the supply of body fluid to the inlet port isremoved, the Laplace pressure of a body fluid meniscus at the inlet portis higher than a threshold pressure of the capillary stop valve, wherebythe capillary stop valve admits flow of the body fluid therethrough; andadmitting a metered volume of body fluid to be transported to a porousmedium arranged in fluid communication with the first channel.

In one embodiment, the capillary stop valve is selected from at leastone of a part of the first channel with altered hydrophilicity; a partof the first channel with changed dimensions.

In one embodiment, the method further comprises collecting the meteredvolume of body fluid in the porous medium acting as a capillary means.

The method facilitates sampling of body fluid by enabling the user tosupply a sufficient amount of body fluid before the body fluid isadmitted to continue to flow through the device for collection in theporous medium.

In a seventh aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport withmeans to disconnect a metered volume from the remaining body fluidbeyond filtration membrane for removing cells, such as red blood cells.The device comprises an inlet section, comprising an inlet port forreceiving a sample of body fluid, the inlet section being configured totransport the sample to a filtration membrane. The device furthercomprises a metering section comprising an extraction chamber arrangedto receive extracted body fluid from the membrane and a meteringchannel. The device also comprises an outlet section configured toreceive, transport, and collect a volume of filtered body fluid from themetering channel in a capillary means. The metering section furthercomprises a pinch-off means configured to separate a metered volume offiltered body fluid in the metering channel from remaining body fluid inthe extraction chamber, wherein the pinch-off means comprises at leastone air vent arranged in a part of the extraction chamber with themaximum height. By means of the air vent, an effective separation of themetered volume from the remaining volume of body fluid is achieved.

In one embodiment, the pinch-off means comprises a pinch-off region influid communication with the at least one air vent, arranged adjacent anentrance to the metering channel, wherein the pinch-off region comprisesa height reducing element with a height lower than the maximum height ofthe extraction chamber. Preferably, the extraction chamber comprises apart with gradually increasing height, a part with a height reducingelement and a part with a maximum extraction chamber height in fluidcommunication with the metering channel. The height reducing elementensures that the pinch-off region has a higher height than the adjacentpart of the extraction chamber, thus reducing the capillary pressure inthe pinch-off region to promote introduction of an air bubble.

In one embodiment, the extraction chamber is substantially wedge-shaped,wherein a roof of the extraction chamber is defined by flat lowersurface of the filtration membrane, and wherein a hydrophilic floor ofthe extraction chamber extends at an acute angle from a contact with thefiltration membrane towards the metering channel. By means of the acuteangle between the filtration membrane and the floor of the extractionchamber, it is possible to achieve a wedge-shaped extraction chamberwhich diverges towards the metering channel, thus enabling gradualfilling of the space between the diverging surfaces, essentially forminga capillary pump. At the same time, it is possible to maintain asubstantially flat, horizontal orientation of the filtration membrane,which facilitates integrating the filtration membrane in a chamberconstruction to protect a blood sample from evaporation andcontamination during plasma extraction. Preferably, the maximum heightof the plasma extraction chamber exceeds the height of the meteringchannel.

In one embodiment, at least one part of the extraction chambersurrounding the pinch-off region has a height lower than the height ofthe metering channel. The surrounding areas of lower height lead to areduction of the capillary pressure in the pinch-off region, thuspromoting introduction of an air bubble

In one embodiment, the device comprises a through-hole in the heightreducing element to prevent liquid from pinning in the extractionchamber.

In one embodiment, the metering section comprises an extraction chamberwith a part with gradually increasing height, a part with the heightreducing element and a part with a maximum extraction chamber heightarranged in fluid communication with the metering channel.

In one embodiment, the device comprises an inlet section comprising aninlet port and a channel system; a filtration membrane configured toseparate plasma from blood, wherein the inlet section and the channelsystem are configured to transport the sample of body fluid to, and todistribute it across the filtration membrane with a stepwise orgradually increasing capillarity from the inlet section to thefiltration membrane along with features as outlined in preceding aspectsof the present disclosure, such as the second aspect.

In one embodiment, the device comprises a metering channel having anoutlet part with a dimensional change configured to cause a fluid frontmeniscus of the separated metered volume of body fluid, when transportedto the outlet section, to assume a shape which substantially conforms tothe surface geometry of the capillary means with features as outlined inpreceding aspects of the present disclosure, such as the third aspect.

In one embodiment, the device comprises an outlet section with aconformable hydrophilic porous bridge element insertable into a bridgecavity in such a way that the bridge cavity is substantially filled withfeatures as outlined in preceding aspects of the present disclosure,such as the fourth aspect.

In one embodiment, the device is a multi-layered device with wedgeshaped extraction chamber wherein a floor of the extraction chamber iscontinuous with a floor of the metering channel and extends at an acuteangle from the lower surface of the filtration membrane, and wherein thefloor of the extraction chamber is inclined with respect to the floor ofthe metering channel to create a slope. The device may be manufacturedusing a multilayer arrangement and a method with features as outlined inpreceding aspects of the present disclosure, such as the fifth aspect.

In one embodiment, the device comprises an inlet part with pre-meteringfunction including visual inspection means and a capillary stop valvewith features as outlined in preceding aspects of the presentdisclosure, such as the sixth aspect.

In an eighth aspect of the present disclosure, there is provided amicrofluidic device configured to sample, meter and collect a meteredvolume of body fluid for analysis by means of capillary transport withmeans to disconnect a metered volume from the remaining body fluidbeyond filtration membrane for removing cells, such as red blood cells.The device comprises an inlet section, comprising an inlet port forreceiving a sample of body fluid, the inlet section being configured totransport the sample to a filtration membrane. The device furthercomprises a metering section comprising an extraction chamber arrangedto receive extracted body fluid from the membrane, a metering channel,and a fluid connector arranged between the extraction chamber and themetering channel and a pinch-off means comprising at least one air ventconfigured to introduce at least one air bubble to separate a meteredvolume. By means of the air vent, an effective separation of the meteredvolume from the remaining volume of body fluid is achieved

In one embodiment, the extraction chamber has a gradually increasingheight to a maximum value, that is less than the height of the meteringchannel.

In one embodiment, the fluid connector has different dimensions than themetering channel preferably such a dimension is selected from one ormore of height, width and/or length.

In one embodiment, the fluid connector has a gradually increasing heightto the maximum height of the metering channel. In a special embodimentof the fluid connector, it is arranged with a height lower than themaximum height at entrance from the extraction chamber and the heightgradually increases to the height of metering channel.

In one embodiment, device has at least one air vent is located in themetering section where the height exceeds the maximum height of theextraction chamber. In one embodiment, the at least one air vent islocated adjacent to, or at the position where the fluid connector meetsthe metering channel. In another embodiment, the at least one air ventis located where the height is at a maximum.

In one embodiment, the at least one air vent is located at the entranceof the metering channel and is configured with an orifice to ambient airwith a cross-sectional area of at least the size of the cross-sectionalarea of the metering channel.

In one embodiment, the fluid connector joins the metering channel at anacute angle or with a curve.

In one embodiment, the extraction chamber is substantially wedge-shaped,wherein a roof of the extraction chamber is defined by flat lowersurface of the filtration membrane, and wherein a hydrophilic floor ofthe extraction chamber extends at an acute angle from a contact with thefiltration membrane towards the metering channel. By means of the acuteangle between the filtration membrane and the floor of the extractionchamber, it is possible to achieve a wedge-shaped extraction chamberwhich diverges towards the metering channel, thus enabling gradualfilling of the space between the diverging surfaces, essentially forminga capillary pump. At the same time, it is possible to maintain asubstantially flat, horizontal orientation of the filtration membrane,which facilitates integrating the filtration membrane in a chamberconstruction to protect a blood sample from evaporation andcontamination during plasma extraction. Preferably, the maximum heightof the plasma extraction chamber exceeds the height of the meteringchannel.

Preferably, extraction chamber, the fluid connector and the meteringchannel have the same hydrophilic floor.

In one embodiment, the device comprises an inlet section comprising aninlet port and a channel system; a filtration membrane configured toseparate plasma from blood, wherein the inlet section and the channelsystem are configured to transport the sample of body fluid to, and todistribute it across the filtration membrane with a stepwise orgradually increasing capillarity from the inlet section to thefiltration membrane along with features as outlined in preceding aspectsof the present disclosure, such as the second aspect.

In one embodiment, the device comprises a metering channel having anoutlet part with a dimensional change configured to cause a fluid frontmeniscus of the separated metered volume of body fluid, when transportedto the outlet section, to assume a shape which substantially conforms tothe surface geometry of the capillary means with features as outlined inpreceding aspects of the present disclosure, such as the third aspect.

In one embodiment, the device comprises an outlet section with aconformable hydrophilic porous bridge element insertable into a bridgecavity in such a way that the bridge cavity is substantially filled withfeatures as outlined in preceding aspects of the present disclosure,such as the fourth aspect.

In one embodiment, the device is a multi-layered device with wedgeshaped extraction chamber wherein a floor of the extraction chamber iscontinuous with a floor of the metering channel and extends at an acuteangle from the lower surface of the filtration membrane, and wherein thefloor of the extraction chamber is inclined with respect to the floor ofthe metering channel to create a slope. The device may be manufacturedusing a multilayer arrangement and a method with features as outlined inpreceding aspects of the present disclosure, such as the fifth aspect.

In one embodiment, the device comprises an inlet part with pre-meteringfunction including visual inspection means and a capillary stop valvewith features as outlined in preceding aspects of the presentdisclosure, such as the sixth aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is now described, by way of example, withreference to the accompanying drawings, in which:

FIG. 1 shows a general outline of a microfluidic device adapted tocollect blood plasma from whole blood by a finger prick, transport andseparate the blood and collect a defined volume of plasma from theblood.

FIGS. 2A-2H show plasma sampling in several consecutive fluid handlingsteps.

FIGS. 3A-3D show a capillary force driven microfluidic device withvolume control of applied sample fluid.

FIGS. 4A-4E show a capillary force driven microfluidic device withvolume control of applied sample fluid with microfluidic featuresintroduced between the indicator window and the connecting capillarysection.

FIGS. 5A-5G show a cross-sectional schematic of a microfluidic deviceusing a capillary stop valve fabricated in lamination technology.

FIGS. 6A-6D show the balancing of capillary pressure in a microfluidicdevice according to an embodiment of the present disclosure.

FIGS. 7A-7G show cross-sectional views of a microfluidic deviceaccording to one embodiment of the present disclosure illustratingdifferent layers that form a pinch-off region.

FIGS. 8A-8C show plan and cross-sectional views of a microfluidic deviceillustrating a pinch-off solution according to one embodiment of thepresent disclosure.

FIGS. 9A-9B show cross-sectional views of a microfluidic deviceillustrating a pinch-off solution according to one embodiment of thepresent disclosure.

FIGS. 10A-10B show cross-sectional views of a microfluidic deviceillustrating a pinch-off solution according to one embodiment of thepresent disclosure.

FIGS. 11A-C show plan and cross-sectional views of a microfluidic deviceillustrating a pinch-off solution according to one embodiment of thepresent disclosure.

FIG. 12 shows a top plan view an embodiment of the microfluidic devicewhich solves the metering accuracy problem by using a fluid connectorwith a venting hole between the extraction chamber and the meteringchannel.

FIG. 13A-13D show top plan views of the microfluidic device comprising afluid connector and four different venting hole designs.

FIGS. 14A-14F show cross-sectional views illustrating steps in amanufacturing method of a microfluidic device according to oneembodiment of the present disclosure.

FIGS. 15A-15F generally demonstrate embodiments of a microfluidic devicehaving a channel system with stepwise increased capillarity that candetermine that a sufficient body fluid volume is introduced.

FIGS. 16A-16F show cross-sectional views of embodiments of the presentdisclosure having a capillary stop valve arranged in fluid communicationwith a pre-metering channel.

FIGS. 17A and 17B show cross-sectional views of an embodiment of amanufacturing method for an outlet portion of a microfluidic device.

FIG. 18 shows top views of an example of bubble formation near at outletin a microfluidic device.

FIG. 19 shows top views of a successful transport of a liquid from achannel to a capillary means according to one embodiment of the presentdisclosure.

FIG. 20 shows a cross-sectional view of a metering channel in amicrofluidic device according to one embodiment of the presentdisclosure.

FIGS. 21A-21B show test results on a metering channel of narrowing widthin a microfluidic device according to one embodiment of the presentdisclosure.

FIGS. 22A-22C show test results on a metering channel of narrowing widthin a microfluidic device according to another embodiment of the presentdisclosure.

FIGS. 23A-23C show test results on a metering channel of narrowing widthin a microfluidic device according to another embodiment of the presentdisclosure.

DESCRIPTION OF EMBODIMENTS

The following section provides detailed descriptions of microfluidicdevices configured to sample and collect a metered volume of body fluidfor analysis by means of capillary transport, according to theembodiments of the present disclosure. In the drawing figures, likereference numerals designate identical or corresponding elementsthroughout the several figures. It will be appreciated that thesefigures are for illustration only and do not in any way restrict thescope of the present disclosure

Example 1—The Microfluidic Device

FIG. 1 shows an exemplary embodiment of a microfluidic device adapted tocollect blood plasma from whole blood by a finger prick, transport andseparate the blood and collect a defined volume of plasma from theblood. In a broad overview the system comprises the following componentswhich are arranged in the direction of flow through the system as shownin FIG. 1 :

-   -   An inlet section 24 comprising    -   an inlet port 4,    -   a channel system 25,    -   a first channel 6, also called pre-metering application channel    -   a second channel 8, also called intermediate channel    -   a third channel 10, also called filtration channel    -   a filtration membrane 12,    -   a metering section 26 comprising,    -   an extraction chamber 14,    -   a vent/pinch-off structure 16,    -   a plasma metering channel 18,    -   an outlet section 28 comprising,    -   an outlet port 21 (with a bridging capillary element 20), and    -   a capillary means 22.

The plasma sampling works in several consecutive fluid handling stepsthat are described in FIGS. 2A-2H. As an overview, the figures show thefollowing: FIG. 2A: the filling of the first, pre-metering application,channel 6 of the inlet section 24; FIG. 2B: the removal of the bloodsupply 30 after the front meniscus 36 of the blood reaches the capillarystop valve 35, leading to forming of a convex rear meniscus 32 of theblood sticking to the inlet port 4; FIG. 2C: Laplace pressure has pushedthe concave front meniscus 36 of the blood liquid across the capillarystop valve 35; FIG. 2D: flow through the second, intermediate, channel 8to the filtration membrane 12, simultaneous filling of the filtrationmembrane, emptying of the pre-metering application channel 6 andinitiation of the plasma extraction; FIG. 2E: filling of the third,filtration, channel 10; FIG. 2F: continuous filtration into theextraction chamber 14; FIG. 2G: filling of the plasma metering channel18; and FIG. 2H: absorption of the metered plasma volume into thecapillary means 22 with a bubble entry at a vent/pinch-off structure 16.

As shown in FIG. 2A, blood 30 is filled via the inlet port 4 into thepre-metering application channel 6. When the pre-metering applicationchannel 6 is filled entirely, the supply of blood to the inlet port ismanually interrupted, thereby metering a defined volume, see FIG. 2B.The intermediate channel 8 transports the blood from the pre-meteringapplication channel 6 towards the filtration channel 10 and thefiltration membrane 12, see FIG. 2C.

The capillary pressure in the intermediate channel 8 thus needs to behigher than the capillary retention pressure that pins the liquid to theinlet port, so that the liquid can be pumped from the pre-meteringapplication channel 6 to the filtration channel 10/filtration membrane12. A higher capillary pressure in the intermediate channel 8 is alsobeneficial for preventing bubbles at the contact of second channel andfiltration membrane 12 where a steep increase in capillary pressure canotherwise introduce air bubbles into the intermediate channel 8. Airbubbles can potentially interrupt the capillary action on the fluid plugthat moves through the system and as a result stop the fluid operations.Once the blood meniscus 32 contacts the filtration membrane/the thirdchannel 10, filling of these two compartments occurs in parallel andaccording to the capillary forces in either of the compartments, seeFIGS. 2D-2E.

Since the third channel 10 and the membrane 12 are arranged in parallel,typically the filtration membrane fills first due to the highercapillary pressure within the filtration membrane. Once the void volumeof the membrane is filled with blood/plasma, the third channel 10starts/continues filling. The filtration membrane 12 has a capillarygradient with pore sizes from several tenths of micrometers on the bloodreceiving side to 2-3 micrometers on the plasma extraction side. As soonas the plasma reaches the lower surface of the filtration membrane 12,the extraction of plasma into the extraction chamber 18 occurs, due tothe high capillary pressure at the intersection of plasma filtrationmembrane 18 and hydrophilic bottom substrate 38, see FIG. 2D. Thediverging space between the membrane 12 and the hydrophilic bottomsubstrate 38 fills gradually with plasma because the capillary pressurein the extraction chamber 14 is substantially higher than the retentionpressure in the pre-metering application channel 6, see FIGS. 2D-2F.

Once the plasma meniscus reaches the inlet of the plasma meteringchannel 18, the plasma continues to flow into the plasma meteringchannel 18 driven by the capillary pressure inside the channel 18, seeFIG. 2G. The capillary pressure inside the plasma metering channel 18needs to be substantially larger than the retention capillary pressurein the pre-metering application channel 6 to allow plasma filtrationthrough the membrane 12. Once the plasma metering channel 18 is filledentirely and the meniscus reaches the outlet port 21, a sudden increasein capillary pressure leads to the absorption of plasma through theoutlet port 21 into the capillary means 22, see FIG. 2H.

Due to the high flow resistance of blood in the filtration membrane,absorption of fluid upstream of the filtration membrane is minimal.Instead, a vent structure/pinch-off structure 16 downstream of thefiltration membrane offers a lower resistance for a bubble entry whichleads to a pinch-off and the metering of the plasma volume. Since thesystem presented is based on the construction of foils which leads toliquid-air interfaces in the downstream capillary system, a bubble entryis possible at several points. Thus, it is important to consider thecapillary retention pressure in the downstream capillary system in orderto have a controlled and repeatable bubble entry that enables thedesired precision in metering the volume of the plasma. Plasmaabsorption through the outlet port continues until the entire plasmametering channel is emptied and the volume is transferred into thecapillary substrate.

Since there is no safety mechanism to prevent a second fill cycle of theplasma metering channel when excessive blood is present at thefiltration membrane, it is crucial to have a well-defined input volume.The input volume is directly correlated with the dead volume of thesystem and the plasma output volume of the system. For this purpose, apre-metering application channel 6 is introduced instead of applyingblood directly on the membrane.

Another reason for introducing a pre-metering application channel 6 isthat the required a total blood volume of blood is approximately 70 μl.Since it is intended that users will apply blood without any measurementdevice such as a pipet, and instead directly from a finger prick, thepre-metering application channel 6 allows collection of severalconsecutive drops and giving feedback to the user about the fill statusof the device. Once sufficient blood has been applied to system, anindicator area will display the successful filling. The pre-meteringapplication channel 6 is also well integrated with the third channelwhich has the purpose of distributing blood homogenously across themembrane and limits evaporation of water from the blood during thefiltration.

Example 2—Pre-Metering

A capillary force driven microfluidic device with volume control ofapplied sample fluid is described generally in FIGS. 3A-3D. The deviceof FIGS. 3A-3D is configured to collect one or more drops at an inletport 40 for transportation into a first, pre-metering application,channel 42 with a pre-metering section/compartment. When thepre-metering section has been filled, a fill indicator 44 confirms thefill status to the user so that the supply of liquid to the inlet port40 can be manually interrupted and a defined volume is trapped in thepre-metering compartment. The pre-metering operation takes place in foursteps: (a) application of liquid to the inlet port 40, (b) capillaryfilling of the pre-metering compartment, (c) reaching of the indicator44, manual read-out, and (d) removal of excess liquid from the inletport 40.

FIGS. 3A-3D illustrate this process. FIG. 3A shows that the liquid isapplied to the inlet port 40. FIG. 3B shows the capillary filling of thefirst channel or pre-metering compartment 42. In FIG. 3C shows that theindicator 44 is reached and, manually read out. In FIG. 3D the excessliquid is removed from the inlet port 40.

Since the manual interruption of fluid supply to the inlet takes placewith a certain delay, it introduces a time dependent overfill of thedefined volume into a second channel or connecting capillary channel,46. This overfill volume depends on the time period between reaching theindicator window 44 and removing the liquid from the inlet port 42, andthe flow rate in the connecting capillary channel 46.

FIG. 4A shows the components of a capillary system including an inletport 50, a first channel 52 (also called pre-metering channel), anindicator window 54 and a second channel 58 (also called connecting orsequent capillary channel). Introducing other microfluidic featuressuitable for capillary driven devices such as a valve or flow reductiongate 56 can help increase the accuracy of the metering. Suchmicrofluidic features can be introduced between the indicator window 54and the second channel 58, to slow down or stop the flow between the twosections, as shown in FIGS. 4B-4E.

FIGS. 4B-4E illustrate the metering of liquid in a capillary systemusing a flow reduction gate or a stop valve 56. The flow reduction gateacts in such a way that the speed of the flow is reduced substantiallyso that, in a given time period (e.g., 3 sec), a smaller volume 57overflows from the pre-metering channel 52 into the second channel 58than without a flow reduction gate, such that the amount of fluidapplied to the capillary system is substantially equal to the meteredvolume of fluid 55 in the pre-metering channel 52. For example, flowreduction gates can be implemented by altering thehydrophilic/hydrophobic properties of the microchannel, adjusting thedimensions of the microchannel, or changing the flow resistance of themicrochannel.

Stop valves such as dissolvable membrane valves or capillary stop valvesbring the flow to a complete halt so that the overfill volume can beminimized. Dissolvable membrane valves can disintegrate when broughtinto contact with a liquid and can stop the flow for a certain time,before opening the fluid communication to the downstream connectingcapillary means. A capillary stop valve acts as a pressure barrier andcan be used to completely interrupt the flow in the capillary systemuntil wetting of the valve occurs or an additional hydraulic pressurepushes the liquid across the pressure barrier. Such a hydraulic pressurecan be introduced in different ways, for example by applying ahydrostatic pressure or by a change in the inlet port conditions, e.g.,a change in Laplace pressure/capillary pressure at the inlet.

The operation of manual removal of excess liquid from the inlet port canbe used to introduce such a change in Laplace pressure that leads to aburst of the stop valve initiating the flow into the second channel.Dimensions and surface properties of the overall capillary system areselected to allow a transport of liquid from the metering section intothe connecting capillary section. Capillary stop valves are not actuallyclosed but create a pressure barrier for the capillary flow which burstsonce a certain pressure is applied to the liquid. One speaks about thebursting of the valve rather than opening of the valve as its notphysically closed but only closed by means of interrupting the capillaryflow. For capillary stop valves, burst pressure is a function of surfaceenergy of the liquid-gas-interface, wettability by the fluid, and thegeometric dimensions of the valve. It therefore can be predefined by anappropriate design of the microfluidic structures.

Consequently, the geometry and/or dimension of the inlet port can beconfigured such that when the supply of body fluid to the inlet port isremoved, the Laplace pressure of a body fluid meniscus at the inlet portis higher than a threshold pressure of the capillary stop valve.

Example 3—Sample Volume Control with a Capillary Stop Valve

FIGS. 5A-5G show an embodiment of a microfluidic device with samplevolume control as generally described in Example 2 using a capillarystop valve 64. 5A-5G show a cross-sectional schematic of a microfluidicdevice using a capillary stop valve fabricated in lamination technology.The device is constructed using structured layers that are laminatedtogether. In FIG. 5A, the cross-section shows an inlet port 60, ametering channel 62, a capillary stop valve 64, the position of anindicator window 66 and a second channel 68. When a drop of liquidcontacts the inlet port 60, liquid is sucked into the metering channel62 of the device until the liquid reaches the capillary stop valve 64(FIGS. 5B-5D). Separation of the excess fluid from the liquid volumeinside the metering channel 62 causes a small amount of liquid to stickto the inlet port 60 outside the metering channel 62.

The curvature of this volume causes the surface tension-induced Laplacepressure on the liquid to push the liquid inside the metering channel 62across the capillary stop valve 64 as indicated by the arrows, by virtueof being higher than the threshold pressure of the capillary stop valve64. The liquid then continues to flow into the second channel 68 becausethe capillary pressure at the front of the liquid flow direction ishigher than the capillary retention pressure at the inlet port (FIGS.5E-5F).

Example 4—Balancing of Capillary Pressure in a Microfluidic Device

FIGS. 6A-6D generally describe balancing of capillary pressure in amicrofluidic device according to the present disclosure. Themicrofluidic device allows for absorption of whole blood into an inletsection, shown as compartment A, 72 and then autonomously filtrates theplasma fraction from whole blood by pumping/transporting the bloodthrough a filtration element (membrane) 74 into a metering section(comprising an extraction chamber and a metering channel) and an outletsection (comprising a capillary means/pump), generally shown ascompartment B, 76 in FIG. 6A. All fluid transport in the device is basedon capillary pressure. The conditions for successful filtration ofplasma require the capillary pressure in Compartment B 76 to be largerthan the retention pressure in the Compartment A 72 so that a fluidtransport occurs from compartment A to compartment B in light of allfrictional forces of the system.

More specifically, the embodiment of the present disclosure comprisesseveral microfluidic elements as described above. Fluid is pumpedthrough the system from the inlet to the outlet forming a fluid plug orcolumn that is pumped through the system using capillary pressure. Toallow the continuous flow of the fluid plug through the system, apressure difference between the capillary pressure at the liquid frontflowing towards the outlet and the capillary pressure at the liquid endtrailing the fluid plug (retention pressure) needs to be given at anytime. The capillary pressure at the meniscus filling into the systemvaries throughout the filling operation and is defined by the contactangle of the interfacing surfaces, the surface tension of the liquid,and the (smallest) channel/feature dimensions. The capillary retentionpressure at the receding end is defined by the same parameters with thedifference that the receding contact angle defines the curvature of theliquid-air interfaces and thus the capillary retention pressure. Whenthe microfluidic device is constructed from laminated layers, thecapillary height is typically much smaller than the channel width; itpredominantly defines the capillary pressure in the different sections.During the application of liquid to the first channel, the liquid is nottrapped in a capillary, but freely available in form of a drop or aliquid reservoir of any shape. This allows filling the precedinglydescribed first channel which has the biggest capillary height in thesystem and thus induces, relatively speaking, the lowest capillarypressure.

Once the application of blood is stopped, the open air-liquid interfacethat trails the fluid plug is formed and is throughout the filling andfiltration operation counteracting the capillary pressure at the liquidfront. To allow a continuous capillary flow of the plug through thedevices, all compartments/channels that follow the liquid front need toinduce a capillary pressure that is substantially larger than thecapillary pressure at the trailing end.

Example 5—Capillary Height Changes

Example 5 is a detailed embodiment of the microfluidic device asgenerally described in Example 4. The microfluidic device in Example 5is fabricated from a stack of structured foils with changes in capillaryheight introduced stepwise, except for the wedge slope. A stepwisereduction in the capillary height can be filled without fluid pinning tothe step. However, a stepwise increase in the capillary height resultsin pinning and formation of a capillary stop, which should be preventedto guarantee a continuous operation of the device. These designrequirements lead to a stepwise decrease in capillary heights throughoutthe system with exception of the plasma extraction chamber, where acontinuous increase of capillary height allows gradually filling of thewedge structure before stepwise decreasing the capillary height again.An example of the operation of the system can be seen in FIGS. 2A-2H;the relevant capillary dimensions are listed in Table 1.

TABLE 1 Device parameters enabling a continuous operation of the deviceas shown in FIG. 2A Compartment Capillary height/Capillary feature sizeFirst channel 750 μm Second channel 300 μm Third channel ~100 μmFiltration membrane Porous gradient from 30 μm to 2 μm Extractionchamber Gradient from 0 μm to 250 μm Plasma metering channel 150 μmCapillary means Pore size 5-10 μm

Examples 6A and 6B below refer to embodiments of the microfluidic devicewith different solutions to pinch-off the metered volume of body fluidin order to transport correctly metered volume for collection in acapillary means at device's outlet.

Example 6A—Metering 1: Pinch-Off Under the Membrane

This embodiment of the present disclosure relates to a pinch-offstructure in a capillary system that allows the separation of a fluidplug into two fluid plugs using capillary force, so that no fluidcommunication between the two plugs occurs. More specifically it allowsthe separation of a well-defined plasma volume from a fluid plugconsisting of whole blood and plasma.

Pinching-off/separating liquids in a capillary driven system requiresthe introduction of an air bubble into the system. Air bubbles can beintroduced to the system at existing liquid-air interfaces such as ventsor other open sections. The wedge structure in the plasma extractionchamber is constructed in a way that due to fabrication constraints, thesealing of the sides of the edge is not possible” However, to allowaccurate metering of plasma, the absorption of plasma below the wedgeand the bubble entry must be controlled. Due to the microfluidicdevice's construction, the parts of the wedge structure that have thehighest capillary height in the plasma extraction system are locateddownstream of the plasma separation membrane, making this a suitablepoint for entering a bubble into the system. In this embodiment of thepresent disclosure, a pinch-off structure is designed that exploits thispoint of relatively low capillary retention pressure in the plasmaextraction chamber and controls where exactly a bubble can enter thecapillary system when the plasma contacts the capillary pump.

FIGS. 7A-7G and 9A-9B both show a pinch-off under the membrane. Pinchingoff occurs once the plasma front reaches the capillary means and theimmediate absorption of plasma from the capillary system is initiated.Since the filtration of plasma through the filter occurs substantiallyslower than the absorption of plasma from the system, the absorptionleads to bubbles growing at the point of least capillary pressure which,in both cases, occur in the section below the filtration membrane. Thisleads to “necking” in the section of highest capillary height until thefluid plug extending between the plasma third channel and the plasmametering channel collapses and the bubble starts to grow in the plasmametering channel. It is an advantage to create necking and pinch-offunder the membrane because the absence of the liquid-solid interfaces onthe left and the right side of the necking region prevents corner flowwhich could otherwise lead to a capillary connection between the twofluid plugs. The corners of a square microchannel have a high capillarypressure which leads to trapping of fluid there that can lead to aremaining connection between the two fluid plugs. Another advantage ofpinching off below the plasma filtration membrane is that, before plasmacan refill the plasma metering channel, the pinch-off region must befilled a second time. Relatively speaking, the refilling occurs ratherslowly since the capillary height here is at its highest level and thus,the capillary pressure is relatively low.

In pinching-off of plasma below the membrane, by narrowing theconnection between the plasma extraction chamber and the plasma meteringchannel, the volume contained in the section designed for pinch-off isreduced. Unwanted absorption of plasma from the section left of thepinch-off region may occur.

The absorption of plasma through the outlet port 21 of the system mayoccur not only from the pinch-off region 84 next to the inlet of theplasma metering channel 18, but also from different areas below themembrane. This unwanted absorption is reduced by the pinch-offstructures 83, 84 shown in FIGS. 7A-7G. The capillary height below thefiltration membrane 81 is reduced by means of a height-reducing element83 in areas where absorption of plasma is undesired and clearly definesa pinch-off region 84, approximately 2 mm×2 mm in surface area, wherethe capillary height has the highest capillary height (in the plasmasystem) of 250 μm. On the right side of the pinch-off region 84, thechannel cover 80 reduces the capillary height to 150 μm and, on the leftside of the pinch-off region 84, the extending structures 83 of thechannel cover 80 reduce the capillary height to less than 150 μm. Inthis way, unwanted absorption of plasma from the wedge-shaped extractionchamber 87 below the membrane 81 is prevented.

In the pinching-off of plasma below the membrane 81, plasma fills fromthe extraction chamber 87 into the plasma metering channel 18. Afterconnecting to the porous plug 89 at the outlet port 21, absorption ofplasma in the plasma metering channel 18 through the outlet port 21occurs and a neck is formed between the plasma extraction chamber 87 andthe plasma metering channel 18. The plasma neck collapses between thethird channel and the plasma metering channel separates the two fluidvolumes.

FIG. 7A schematically shows a longitudinal cross-sectional view cutthrough lines G-G of an embodiment of the microfluidic device with apinch-off region 84, while FIGS. 7B-7G show the transversal cut-throughlines A-A, B-B, C-C, D-D, E-E, and F-F, respectively. FIG. 7F shows theoverlap between the bottom 82 of the plasma metering channel 18 and theroof 80 of the plasma metering channel 18, defining the capillary height88 of the plasma system. The pinch-off region 84 is defined by reducingthe capillary height upstream (left in FIG. 7A) and downstream (right inFIG. 7A) of the pinch-off region 84. The pinch-off region has opensidewalls 86 creating a liquid-air interface that is beneficial forbubble entry and prevents corner flow.

Pinch-off under the membrane according to the design shown in FIGS.7A-7G occurs as follows:

Before wetting the porous plug 89 at the outlet 21, the pinch-off region84 below the membrane 81 is filled with plasma. The wetting of theporous plug 89 leads to absorption of plasma from the pinch-off region84 and a neck is formed. Further absorption of plasma from the neckingregion leads to a collapse of the neck and disconnects the fluid in theplasma extraction chamber 87 from the fluid in the plasma meteringchannel 18. A bubble then enters the plasma metering channel 18 as thefluid in the channel 18 is absorbed through the outlet port 21 of thedevice. Refilling of the pinch-off region occurs from the plasmaextraction chamber 87 as plasma filtration continues.

FIG. 9A shows a longitudinal cross-sectional view of an embodiment ofthe metering 1 solution where the extraction chamber 102 issubstantially wedge-shaped and with a horizontally arranged filtrationmembrane 100 as a roof and slope 104 formed by a hydrophilic floor 106extending at an acute angle from a contact with the filtration membranetowards the metering channel 108. FIG. 9B shows a transversecross-sectional view taken along line A-A and illustrates filling ofplasma 109 in the pinch-off region prior to pinch-off throughintroduction of an air bubble.

Example 6B—Metering 2: Using a Pinch-Off Structure Inside the MeteringChannel

As an alternative to the metering 1 solution shown in FIG. 9A-B, inFIGS. 8A-C and FIGS. 10A-B the capillary height H1 under the membrane 98can be reduced to be smaller than the height H2 of the metering channelthus preventing unwanted absorption of plasma below the membrane, butinstead facilitating the formation of a bubble inside the meteringchannel 90 at the location of the vent 92. This is achieved by shiftingthe starting point of the slope 96 further outside of the membrane 98 todefine the wedge-shaped extraction chamber which is formed betweenhydrophilic channel floor 93 and the filtration membrane 98, asillustrated in FIGS. 8B and 10A. This enables introduction of a bubbleby placing a vent structure 92 in the metering channel 90. Thisembodiment of the present disclosure relates to using a pinch-offstructure inside the metering channel 90.

In FIG. 10A the maximum height H1 of the extraction chamber is less thanthe height of the metering channel H2, thus rendering H2 the highestcapillary height in the metering channel 90. As the pinch-off occurs,this will cause a bubble to be pulled at the location of the vent 92adjacent to the entrance to the metering channel 90 upon contact of thefluid in the metering channel 90 with a capillary means 94 at theoutlet. FIG. 9B shows a transverse cross-sectional view taken along lineA-A and illustrates filling of plasma 109 in the pinch-off regionadjacent the air vent 92 prior to pinch-off through introduction of anair bubble

FIGS. 11A-11C show an alternative embodiment of a microfluidic devicewith pinch-off inside the metering channel wherein the metering channelis non-straight, e.g., substantially Z-shaped. FIG. 11A shows a top planview of the microfluidic device with a filtration membrane 110 arrangedabove the extraction chamber similar to the embodiment in FIG. 8A. Avent 92 is arranged adjacent the metering channel 90 at a location wherethe metering channel 90 makes a 90-degree bend. This placement increasesthe surface area of the liquid-air interface at the vent 92, as will bedescribed more in detail below. FIGS. 11B and 11C show cross-sectionalviews taken along lines A-A and B-B, respectively, illustrating thestructures of the microfluidic device.

Example 7—L-Shaped Metering Channel

Testing of various prototypes has revealed that it was necessary tocarry out the bubble pinch-off as fast as possible, i.e., as close aspossible to the position where the extraction chamber meets the meteringchannel to avoid absorption of surplus plasma from under the membrane.The unwanted absorption of plasma from under the membrane depended onthe blood properties, i.e. hematocrit levels, which was not acceptable.Unwanted absorption of plasma is a result of the resistance (or lackthereof) exhibited by the membrane compartment. This is generated byfactors such as clogging of pores in the membrane with red blood cells(RBCs) (hence hematocrit dependent), interactions between membrane,channel bottom layer (the slope) and membrane etc.

Furthermore, while this system works adequately for blood with ahematocrit level of 55 or 45, it has been observed that for hematocritlevels of 35 or below, some of the plasma does not follow the desiredflow path to the outlet, and thus the metering of the plasma is nolonger accurate. The lower the hematocrit the fewer red blood cells toclog the membrane hence the lower resistance in the membrane. Thisresulted in that plasma flows very fast from the plasma extractionchamber into the metering channel and the bubble has difficulties inpinching off.

By testing prototypes, it was found that one way of solving the meteringaccuracy problem was to use a fluid connector 124 between the extractionchamber 122 below the membrane 120 and the metering channel 128, asgenerally depicted in the embodiment of FIG. 12 . The embodiment of FIG.12 has a venting hole 126 that allows for introducing a bubble topinch-off as close as possible to the fluid connector 124 and perform apinch-off as quickly as possible after introducing the bubble in thesystem. This reduced the surplus HCT dependent flow from the membranecompartment. It has also been discovered that the geometry of theventing hole in the L-shaped metering channel plays a role in how easilya bubble can be introduced into the system. For a bubble to beintroduced at the venting hole, Fp<Fc, where Fp is the capillary forceacting on the liquid at the venting hole 126 and Fc is the capillaryforce acting on the liquid at the outlet 129. If Fp>Fc a bubble will bepulled from the outlet 129 instead. For this reason, it is desired thatFp is as low as possible. The factors contributing to Fp are pinning ofthe fluid to edges of the venting hole 126, capillary forces and theliquid-air interface of the vent amongst others. It was empiricallydemonstrated that the larger the liquid-air interface, the easier it isto introduce a bubble. This is believed to be the result of the tendencyof a liquid to shrink into the minimum surface area possible due tosurface tension.

FIGS. 13A-13D show four different venting holes 126 designs where 13Ahas the smallest liquid-air interface 127 a, 13B with a slightly largerliquid-air interface 127 b substantially corresponding to the dimensionof the metering channel 128, 13C with a larger oblique liquid-airinterface 127 c, and finally 13D with the largest non-straightliquid-air interface 127 d. In design A, the liquid needs to expand froma small liquid-air interface to a larger one (the cross section of themetering channel). In B, it goes from the same cross-sectionalliquid-air interface throughout the bubble formation. In both C and Dhowever, liquid-air interface at the vent is larger than the channelcross section resulting in that less force is required for bubbleintroduction in the channel.

Example 8—Method of Production

One embodiment of the microfluidic device relates to enabling a slope ina microfluidic substrate in order to generate a height gradient.

Initiating plasma flow from a plasma extraction membrane requires aforce which can be exhibited passively (capillary driven) or actively byapplying an external force. One way of establishing capillary flow isplacing a plasma extraction membrane at an angle across a microchannelopening. The membrane then forms an acute angle between the channelbottom and roof creating a capillary force driven flow under themembrane which is transported into the microchannel. The time it takesfor a specific blood volume to pass through the membrane and extract itsplasma is in general in the range of minutes and is depending on thehematocrit of the blood, hence it can also vary. Given this timespan, itis necessary to protect the blood sample from evaporation during theextraction. From a usability point of view, it is also necessary toprotect the blood volume from contamination. Consequently, for enablinga product using microfiltration-based plasma filtration, the filtrationmembrane needs to be integrated in a chamber construction.

From a microfabrication point of view, integrating an uneven object likea plasma membrane placed at an angle into a chamber structure ischallenging as it creates steps of different heights over a surfacewhich are difficult to seal off liquid tight.

Generally, the plasma extraction membranes are constructed from flexiblepolymer materials or cotton fibers resulting in that the wedgeconstruction offers no rigid support for subsequent layers to build on.For integration in a chamber, it is preferred that the plasma extractionmembrane exhibits a horizontal surface. For enabling this, it isrequired to create a slope on the microfluidic substrate to create thewedge structure between channel and membrane.

The common industrially scalable manufacturing technologies such asmicro injection molding, R2R hot embossing, were considered, as well asless scalable additive methods such as 3D printing, dispensing andcasting. However, these methods were dismissed as inadequate. Firstly,the existed difficulties finding a manufacturer capable of producing atool with a slope for injection molding or hot embossing or casting.Secondly, none of these methods were capable of producing the requiredhydrophilic surface of the slope. For these methods a hydrophilictreatment would be a requirement adding further complexity to themanufacturing method. Lastly, none of these methods were scalable. Toovercome these challenges, a solution for creating the slope wasdeveloped.

In particular, Example 7 demonstrates a method suitable to produce aheight gradient in microfluidic channels in devices using foilsubstrates and lamination-based manufacturing technologies. The use ofthin foils allows for bending the foil substrate or parts of it out ofthe plane to enable a slope that can be incorporated in a microfluidicsubstrate.

By isolating a part of the microfluidic bottom substrate, attaching itto a bottom substrate as in A and placing the other end of the isolatedstructure on top of a support structure as in B, a slope can be producedin the bottom substrate of the channel.

FIGS. 14A-14F show cross-sectional views illustrating steps in amanufacturing method according to one embodiment of the presentdisclosure, for producing a plasma sampling system in the form of amicrofluidic device. In order to incorporate the plasma extractionmembrane in a chamber to prevent the sample from evaporating, protect itfrom contamination and enable a pre-metering of the sample, it isnecessary to have the plasma extraction oriented horizontally ratherthan in a slope as shown in WO 2016/209147 A1, the contents of which areincorporated herein in its entirety. In order still to have a wedgeformed the between membrane and channel bottom, the suggested method forcreating a slope in a channel was implemented.

FIG. 14A shows a first layer in the form of a bottom substrate foil 130prepared with a first opening 131 for an extraction chamber extendingbetween points a and b, and a second opening 133 at point c foraccommodating a capillary means such as a paper substrate at an outlet.

FIG. 14B shows a second layer in the form of a support structure 132assembled on the first layer creating a plateau on the bottom substrate130 adjacent point a of the first opening 131. The support structure 132could be made out of dsPSA, dispensed or screen-printed polymer.

FIG. 14C shows a third layer in the form of a hydrophilic floor layer134 assembled on the first and second layers. The third layer isintended to constitute a continuous floor of the extraction chamber aswell as a metering channel in fluid communication with the extractionchamber, in one piece. To this end, the part forming the floor of theextraction chamber is inclined with respect to the floor of the meteringchannel such that a slope 135 is created. The free end of the slope 135is supported on and attached to the support structure 132 adjacent pointa, whereas the remaining part of the floor layer 134 is attached to thebottom substrate 130 adjacent point b and extending towards and at leastpartially covering the second opening 133 adjacent point c. Thus, theslope extends across the first opening 131 between points a and b. Thefloor layer 134 may have an opening which is aligned with the secondopening 133 of the bottom substrate 130 when the two are assembled, thusforming an outlet port 142. The third layer may be composed of ahydrophilic foil material facing up and an adhesive layer facing down.

In one embodiment, the slope 135 is formed by a slot in the floor layer134, delimiting a tongue portion. The slot can be substantially C-shapedto delimit a substantially circular or substantially square tongueportion on three sides. In this case, the free end of the tongue portionis supported on the support structure 132 adjacent point a, while thepart of the floor layer 134 adjacent the free end of the tongue portionis attached to the bottom substrate 130, as shown on the left side ofFIG. 14C.

FIG. 14D shows a fourth layer in the form of a channel structure layer138 assembled on the third layer 134. The channel structure layer 138comprises an opening to accommodate the support structure 132 and theinclined slope 135 constituting the floor of the extraction chamber, aswell as a slot which forms side walls of the metering channel. Thefourth layer may be made from a double-sided PSA tape cut with a channelstructure and membrane chamber opening.

FIG. 14E shows a fifth layer in the form of a channel cover layer 140assembled on the fourth layer. The channel cover layer 140 comprises anopening substantially corresponding to the size of the extractionchamber 137 and may be arranged such that a part thereof is attached tothe support structure 132 adjacent the free end of the slope 135 of thefloor layer 134. The fifth layer may be composed of a hydrophilicsurface facing down and adhesive surface facing up. The hydrophilicsurface constitutes the roof of the metering channel and the adhesivesurface enables attachment of additional layers on top of the channelcover layer 140.

FIG. 14F shows the five-layer construction now providing a flat topsurface facilitating subsequent assembly of a filtration membrane 141and additional structures 148 to form a chamber around the filtrationmembrane 141. A wedge-shaped extraction chamber 137 is created betweenthe floor layer 134 and the plasma extraction/filtration membrane 141due to the slope 135 extending between points a and b. The extractionchamber 137 reaches its maximum height at the entrance 139 to themetering channel, adjacent point b.

Further embodiments of this invention involve increased use andexploration of height gradients in microfluidic systems. Such furtherembodiments are to be used in the applications mentioned in thebackground. For example, the sloped channel can be filled with either aliquid or a hydrogel to study diffusion effects.

FIGS. 15A-15F show a generalized microfluidic device with an inlet port152, a first, pre-metering application, channel 154 and a second,intermediate, channel 156. A drop of body fluid 150 is applied to theinlet port and admitted to be transported by the capillarity of thefirst channel 154. When the fluid is transported to a means for visualinspection 155 such as an indicator window, the fluid is observed by theuser who then removes excess fluid from the inlet port 152 whereby thefluid is admitted to be further transported, for example to any porousmedium for collection, analysis or further processing. The device mayfurther comprise a third, filtration, channel 158 with a highercapillarity than the pre-metering application channel 154 and theintermediate channel 156. Herein, the filtration channel 158 is arrangedin fluid communication with a porous plug 159 that for example can be afiltration membrane or a lateral flow medium.

FIGS. 16A-16F show a microfluidic device with a capillary stop valve 166arranged in fluid communication with a metering channel 164. FIG. 16Aand FIG. 16B shows a how a drop of body fluid 160 is applied to theinlet port 162 and transported as a fluid flow by capillarity in a firstchannel 163 (also called application chamber) towards the meteringchannel 164. In FIG. 16C the fluid flow front has arrived at thecapillary stop valve 166 which can be inspected by the user by thevisual inspection means 168. In FIG. 16D the user removes the body fluid160 from the inlet port 162 whereby a fluid column is formed whichestablishes a sufficient pushing force to overcome the capillary stopvalve 166, so the fluid column is admitted to further proceed to theporous plug 167 (FIGS. 16 D & E) and be collected in the capillary means169 (FIG. 16F).

Example 9—Manufacturing Outlet Portion

A method for connecting a microfluidic channel to a paper substratewhich enables transferring of a liquid in the channel onto the paper isnow disclosed; this method is compatible with mass manufacturing.

This method involves using a porous, but highly compressible materialwhich can conform to the shape of the outlet hole and be compressed toallow for the paper substrate to contact the adhesive on the bottom ofthe channel substrate. The porous material could be dispensed into thehole or be placed over the hole and then compressed into it. Materialsthat could be used for the porous plug include, for example, micro paperpulp, micro fibrillated cellulose (MFC), open-cell hydrophilic polymerfoams or a highly compressible glass fiber web.

FIGS. 17A and 17B show cross-sectional views of an embodiment of amanufacturing method using glass fiber web, before and after assembly.In FIG. 17A, an outlet of a microfluidic device is shown, at the distalend of a plasma metering channel 170 terminating in an outlet hole 171,forming a cavity 172. A porous plug 174 made of glass fiber material isarranged adjacent the outlet hole 171 to form a bridge element betweenthe metering channel 170 and capillary means such as a paper substrate176. The porous plug 174 has been cut smaller than the paper substrate176 to allow for bonding between an adhesive surface 178 on theunderside of the floor layer of the microfluidic device and the papersubstrate 176, but larger than the outlet hole 171 to ensure no gaps canbe generated between porous plug 174 and the outlet 171.

Referring now to FIG. 17B, the porous plug 174 is inserted into andsubstantially fills the cavity 172 by application of a pressure to theporous plug 174 and the paper substrate 176. To this end, the porousplug 174 is arranged to conform to the shape of the cavity 172. In oneembodiment, the porous plug 174 is formed of a compressible materialwhich allows it to enter the outlet hole 171 and then expand into thecavity 172. As a result of the applied pressure, a compression of glassfibers adjacent to the outlet hole 171 is shown as a thick line.

In another embodiment, a dispensable material is dispensed into theoutlet hole 171 and then allowed to set to form the porous plug 174. Thevolume of the material will adapt to arrive at the same result, i.e., abridge element which conforms to the shape and substantially fills thecavity 172 while ensuring that no air gaps could form in the outletgeometry. At the same time allowing for adhesion between the papersubstrate 176 and the bottom of the microfluidic device.

The particular design of the system solves several challenging issues intransferring a liquid from a channel to a paper: The use of a materialwhich is highly compressible or can be dispensed, reduces the need forhigh precision-cutting and placement of the porous plug into the outlethole. Consequently, this allows for application of the solution inautomatized high throughput manufacturing. In this example, the glassfiber material and the 6 mm paper disk were punched out with diametersof 3 mm and 6 mm, respectively. The two discs were placed on the 2 mmdiameter outlet hole and only aligned by the eye. The solution does alsonot need any PVA coating on the collection substrate which reduces costof the technology.

Example 10—Straightening the Meniscus

The different flow profiles of a liquid in a rectangular microchanneldepend on channel geometry and the interaction between channel materialand liquid. The flow in the channels of the microfluidic device of thepresent disclosure is shear driven flow. Corner flow is influenced bycorner angle and wetting contact angle. In order to maintain continuousflow in a microchannel, bubble formation needs to be avoided.

FIG. 18 shows an example of bubble formation using a porous plug at theoutlet. The liquid meniscus impacts with the porous plug at the bottompart of it causing a bubble to expand at the upper part of the plug. Inthis embodiment of the present disclosure the porous plug was made ofglass fiber web.

FIG. 18 shows the sequence of events when a liquid meniscus in a channelencounters a porous plug entered into the outlet hole of the channel.Due to the mismatch in shape of the meniscus and porous plug, the firstimpact takes place at the bottom part of the plug causing air to bedrawn into the system and a bubble is formed which then expands into thechannel. Since the goal is to transport the liquid from the channel tothe paper, the presence of the bubble threatens to block and cut off theflow and, if the liquid in the channel to be emptied is metered, thiswill reduce the metered volume by its presence.

Bubble formation can be avoided by adapting the shape of the fluid frontmeniscus to the geometry of the capillary means such that the shapes atthe interface match each other.

To ensure that no bubbles are generated during the interaction betweenthe porous plug and the liquid meniscus, it is foreseen to reduce thewidth of the metering channel. The reduction in width causes the liquidmeniscus to go from a convex shape to a substantially straight, planarshape. At the same time, the curvature of the interface of the porousplug has also been straightened through the reduction in channel width.The result is that the shapes of the interfaces match each other.

Referring now to FIG. 19 , there is shown a successful transport of aliquid from a channel 190 to a paper substrate 194 using the proposedinvention. This example uses a 3 mm diameter glass fiber material as aporous plug 192 and a 6 mm diameter paper disc substrate 194. In a firstregion, the channel 190 has a width of about 2 mm, in a second regionthe width of the channel 190 gradually narrows and in a third region thechannel 190 has a width of about 1 mm.

The narrowing at the outlet allows for re-shaping of the liquid meniscusinto a straight liquid front which facilitates control of the impactwith the porous plug and prevents bubble formation at impact between thetwo medias. The solution using the glass fiber disc was proven robust infurther investigations and was successfully evaluated for plasmaextraction and metering of whole blood in the hematocrit range of 30-55HCT.

Furthermore, this solution is readily applicable to other downstreamsystems for integration in point of care and rapid diagnostic testsystems.

FIG. 20 shows a cross-section of the metering channel in the presentlydisclosed microfluidic device. The top and bottom material is composedof a hydrophilic foil and the channel sidewalls of a double-sidedpressure sensitive adhesive tape (dsPSA).

In this microfluidic system, the channel material (bottom, top andsidewalls) and cutting method creating the side wall characteristics(roughness, wettability after cutting, corner angle) affect the shape ofthe meniscus. The shape of the meniscus is critical at the time ofconnecting with the glass fiber bundle at the outlet to avoid pulling abubble.

Different combinations of these parameters were tested, and the optimalcombination for obtaining the shape of a meniscus that matches the shapeof the outlet fiber bundle at the timepoint where the two connect toobtain bubble-free connection was discovered.

The following parameters were tested:

-   -   Hydrophilic material for top and bottom (A<B<C in degree of        hydrophilicity)        -   A. PCS        -   B. Tesa        -   C. Coveme polyester film    -   Sidewall material; (different double-sided pressure sensitive        adhesive tapes)        -   D. Tesa        -   E. Produced in-house        -   F. PCS        -   G. AR Care        -   H. AR Seal    -   Cutting method        -   I. Knife plotting        -   J. Laser A        -   K. Laser B    -   Outlet narrowing width        -   L. 1 mm        -   M. 0.7 mm        -   N. 0.4 mm

Results:

FIGS. 21A & 21B show a test using a channel of 2 mm width with a gradualnarrowing, resulting in a width of 1 mm in the region adjacent theoutlet. The bottom and top materials of the channel are made of Coveme,and the sidewalls of AR Seal. A Laser A cutting method was used. FIG.21A shows a substantially planar meniscus in the 2 mm wide meteringregion; in FIG. 21B a convex meniscus is generated in the region of 1 mmwidth after narrowing.

FIGS. 22A & B show a test using a channel of 2 mm width with a gradualnarrowing, resulting in a width of 1 mm in the region adjacent theoutlet. The channel's bottom and top material are made of Coveme, andthe sidewalls of in-house produced double-sided pressure sensitiveadhesive tape. A knife plotting cutting method was used. FIG. 22A showsa concave meniscus in metering channel in the 2 mm wide metering region,and in FIG. 22B the meniscus is still concave after reduction of channelwidth to 1 mm. In FIG. 22C the meniscus is planarized after furtherreduction of the channel width to 0.4 mm in the region adjacent theoutlet.

FIG. 21A-B and FIG. 22A-C show how different menisci can be producedusing the same hydrophilic foil Coveme in combination with two differentcutting methods and materials. The resulting meniscus in the narrowingin FIG. 21B with its convex nature does not allow for bubble freeconnection to the fiber bundle due to its mismatching surface. A bubblefree connection would not appear in the 2 mm region with a straightmeniscus either. In FIGS. 22A-C, it was required to reduce the width ofthe outlet narrowing to 0.4 mm (FIG. 22C) to planarize the plasmameniscus and adapt it to the fiber bundle surface. However, the width ofthe narrowing was too small for allowing effective contact and emptyingthrough the fiber bundle.

FIGS. 23A-C show the solution is implemented in the presently disclosedmicrofluidic device. FIGS. 23A-C show a test using a channel of 2 mmwidth with a gradual narrowing, resulting in a width of 0.7 mm in theregion adjacent the outlet. The bottom and top material of the channelis made of Tesa, and the sidewalls of in-house produced double-sidedpressure sensitive adhesive tape. A Laser B cutting method was used. InFIG. 23A a concave meniscus has formed in the metering channel andwobbling a bit as it proceeds through the metering channel. In FIG. 23Bthe meniscus is planarized and wobbles less after reduction of channelwidth to 0.7 mm, while in FIG. 23C, after further advancement themeniscus has become straight and adapted to the glass fiber bundle,allowing for bubble free connection and emptying.

Embodiments of a microfluidic device configured to sample, meter andcollect a metered volume of body fluid for analysis by means ofcapillary transport and corresponding methods according to the presentdisclosure have been described. However, the person skilled in the artrealizes that the embodiments can be varied within the scope of theappended claims without departing from the inventive idea.

All the described alternative embodiments above or parts of embodimentscan be freely combined without departing from the inventive idea as longas the combination is not contradictory.

List of Reference Numbers:  2 microfluidic device  4 inlet port  6 firstchannel (pre-metering application channel)  8 second channel(intermediate channel)  10 third channel (filtration chamber)  12filtration membrane  14 extraction chamber  16 vent structure/pinch-offstructure  18 plasma metering channel  20 porous bridge element  21outlet/outlet port  22 capillary means  24 inlet section  25 channelsystem  26 metering section  28 outlet section  30 body fluid (blood) 32 fluid rear meniscus  35 capillary stop valve  36 fluid frontmeniscus  38 hydrophilic bottom substrate  40 inlet port  42 firstchannel (pre-metering application channel)  44 indicator window  46second channel (connecting capillary channel)  50 inlet port  52pre-metering channel  54 indicator  55 metered volume  56 flow reductiongate (capillary stop valve)  57 overflow volume  58 second channel(sequent channel)  60 inlet port  62 first channel (pre-meteringapplication channel)  64 capillary stop valve  66 indicator window  68second channel (sequent channel)  72 compartment A  74 filtrationelement  76 compartment B  80 channel cover  81 filtration membrane  82hydrophilic floor  83 height reducing element  84 pinch-off structures 85 slope  86 open sidewalls  88 capillary height  89 porous plug  90metering channel  92 vent  93 hydrophilic channel floor  94 porous plug 96 slope  98 filtration membrane 100 filtration membrane 102 extractionchamber 104 slope 106 hydrophilic floor 108 metering channel 109 plasma110 filtration membrane 120 filtration membrane 122 extraction chamber124 fluid connector 126 venting hole 127a liquid-air interface 127bliquid-air interface 127c liquid-air interface 127d liquid-air interface128 metering channel 129 outlet 130 first layer (bottom substrate foil)131 first opening a-b 132 second layer (support structure) 133 secondopening c 134 third layer (hydrophilic floor) 135 slope (floor ofextraction chamber) 136 floor of metering channel 137 extraction chamber138 fourth layer (channel structure) 139 entrance to metering channel140 fifth layer (channel cover) 141 filtration membrane 142 outlet port148 chamber structure 150 body fluid 152 inlet port 154 first channel(pre-metering application channel) 155 visual inspection means 156second channel (intermediate channel) 158 third (filtration) channel 159porous plug 160 body fluid 162 inlet port 163 first channel(pre-metering application channel) 164 second channel (sequent channel)166 capillary stop valve 167 porous plug 168 visual inspection means 169capillary means 170 metering channel 171 outlet hole 172 cavity 174porous plug 176 paper substrate 178 adhesive surface 190 channel 192porous plug 194 paper disk substrate

1. A microfluidic device configured to sample, meter and collect a metered volume of body fluid for analysis by means of capillary transport, wherein the device comprises: an inlet section, for receiving a sample of body fluid, the inlet section comprising an inlet port; a metering section configured to receive body fluid from the inlet section and comprising a metering channel, wherein the metering section is arranged to separate a metered volume of body fluid filled in the metering channel; and an outlet section configured to receive and transport the separated metered volume of body fluid for collection in a capillary means having a predetermined surface geometry, wherein the metering channel has an outlet part with a dimensional change configured to cause a fluid front meniscus of the separated metered volume of body fluid, when transported to the outlet section, to assume a shape which substantially conforms to the surface geometry of the capillary means.
 2. The device according to claim 1, wherein the dimensional change comprises a reduction in width and/or height of the metering channel.
 3. The device according to claim 2, wherein a distal end of the outlet part of the metering channel adjacent the capillary means has a constant width which is smaller than the width of the metering channel.
 4. The device according to claim 3, wherein the outlet part of the metering channel has a first part with a gradual reduction in width and second part with a constant width which is smaller than the width of the metering channel.
 5. The device according to claim 1, wherein the surface geometry of the capillary means at an interface surface with the fluid front meniscus is curved or substantially planar.
 6. The device according to claim 1, wherein the capillary means comprises a bridge element arranged in fluid communication with the outlet part of the metering channel and a paper substrate connected to the bridge element.
 7. The device according to claim 6, wherein the bridge element is a hydrophilic porous element with an average pore size smaller than the smallest dimension of the metering channel.
 8. The device according to claim 7, wherein the bridge element, is made from a material selected from at least one of micro paper pulp, micro fibrillated cellulose, an open cell hydrophilic polymer or a highly compressible glass fiber web.
 9. The device according to claim 6, wherein the surface geometry of the bridge element at an interface surface with the fluid front meniscus is curved or substantially planar.
 10. The device according to claim 1, further comprising a filtration membrane configured to separate selected cells from the body fluid, wherein the inlet section is configured to transport the sample of body fluid to, and to distribute it across the filtration membrane and wherein the metering section comprises an extraction chamber configured to receive body fluid from the filtration membrane and to transport the received body fluid to the metering channel.
 11. The device according to claim 10, further comprising a pinch-off means configured to separate the metered volume of body fluid, wherein the pinch-off means comprises at least one air vent arranged in a part of the extraction chamber with a maximum height.
 12. The device according to claim 11, wherein the pinch-off means comprises a pinch-off region in fluid communication with the at least one air vent, the pinch-off region being arranged in the part of the extraction chamber with the maximum height and surrounded by areas with lower height.
 13. The device according to claim 12, wherein at least one part of the extraction chamber surrounding the pinch-off region has a height lower than the height of the metering channel.
 14. The device according to claim 10, wherein the metering section comprises a fluid connector extending between the extraction chamber and the metering channel, and an air vent.
 15. The device according to claim 14, wherein the air vent is arranged adjacent to, or at the position where the fluid connector meets the metering channel.
 16. The device according to claim 15, wherein the air vent is arranged at the entrance of the metering channel and is configured as an orifice to ambient air with a cross-sectional area equal to or greater than the size of the cross-sectional area of the metering channel.
 17. The device according to any claim 14, wherein the fluid connector has a different dimension than the metering channel, the dimension being selected from one or more of height, width and length.
 18. The device according to claim 17, wherein the fluid connector has a gradually increasing height towards the entrance of the metering channel.
 19. The device according to any one of claim 14, wherein the maximum height of the extraction chamber is lower than the height of the metering channel.
 20. A method for sampling, transporting and collecting a metered volume of body fluid for analysis by means of capillary transport from an inlet to a capillary means of a microfluidic device, the method comprising the steps of: applying a sample of body fluid to an inlet port of the device and transporting the body fluid, optionally through a filtration membrane, to a metering channel; admitting the metering channel to transport the sample of body fluid to an outlet section comprising a capillary means having a predetermined surface geometry; receiving the metered fluid in the capillary means and separating a metered volume of body fluid from the remaining sample volume by introducing at least one air bubble at a point of the device upstream of the metering channel exhibiting low capillary pressure; and collecting the metered volume of body fluid in the capillary means, wherein an outlet part of the metering channel comprises a dimensional change which causes a fluid front meniscus of the separated metered volume of body fluid, when transported to the outlet section, to assume a shape which substantially conforms to the surface geometry of the capillary means. 